Pleuromutilin derivatives

ABSTRACT

Pleuromutilin compounds of the formula: 
                 
 
are of use in anti-bacterial therapy.

The present invention relates to novel compounds, to processes for theirpreparation, to pharmaceutical compositions containing them and to theiruse in medical therapy, particularly antibacterial therapy.

Pleuromutilin, the compound of formula (A), is a naturally occurringantibiotic which has antimycoplasmal activity and modest antibacterialactivity. Mutilin and other compounds with a free OH at C-14 areinactive. The impact of further modification at C-14 on the activity ofpleuromutilin has been investigated (H. Egger and H. Reinshagen, J.Antibiotics, 1976, 29, 923). Replacing the hydroxy group of the glycolicester moiety at position 14 by another O, S or N-linked group was foundto improve anti-microbial activity. Thus, introducing adiethylaminoethylthio group gives the compound of formula (B), alsoknown as Tiamulin, which is used as a veterinary antibiotic (G.Hogenauer in, Antibiotics, Vol. V, part 1, ed. F. E. Hahn,Springer-Verlag, 1979, p.344).

In this application, the non-conventional numbering system which isgenerally used in the literature (G. Hogenauer, loc. cit.) is used.

WO 97/25309 (SmithKline Beecham) describes further modification of theacyloxy group, disclosing inter alia 14-O-carbamoyl (R^(a)ONR^(b)CO₂—and R^(a)CONR^(b)CO₂—) derivatives of mutilin in which R^(a) may have arange of values, including saturated or unsaturated optionallysubstituted (cyclic) hydrocarbon, or an optionally substituted aryl orheterocyclic group and R^(b) is a selected from a variety of monovalentgroups.

WO 98/05659 (SmithKline Beecham) describes further 14-O-carbamoylderivatives of mutilin in which the N-atom of the carbamoyl group isacylated by a group which includes an azabicyclic moiety.

WO 99/21855 (SmithKline Beecham) describes further derivatives ofmutilin or 19,20-dihydromutilin, in which the glycolic ester moiety atposition 14 is replaced by the group R²(CH₂)_(m)X(CH₂)_(n)CH₂COO— inwhich R² is a non-aromatic mono- or bicyclic group.

WO 00/27790 (SmithKline Beecham) describes C-14 spirocyclic,acylcarbamate, heteroaryalkyl carboxylate or arylalkoxyalkyl carboxylatederivatives of mutilin or 19,20-dihydromutilin.

WO 00/37074 (SmithKline Beecham) describes further derivatives ofmutilin or 19,20-dihydromutilin having a heteroaryl acetate substituentat the C-14 position.

WO 00/3287 (SmithKline Beecham) describes further derivatives of mutilinor 19,20-dihydromutilin having an isoxazoline carboxylate substituent atthe C-14 position.

WO 01/14310 (SmithKline Beecham) describes further derivatives ofmutilin or 19,20-dihydromutilin having a β-ketoester substituent at theC-14 position.

In addition, 19,20-dihydro-2a-hydroxy-mutilin is described by G. Schulzand H. Berner in Tetrahedron, 1984, vol. 40, pp 905-917, and a number ofC-14 ether, carbamate, amide and urea derivatives of mutilin or19,20-dihydromutilin are described by Brooks et al. in Bioorg. Med.Chem, 2001, vol. 9, pp1221-1231.

The present invention is based on the unexpected discovery that certainnovel C-14 oxycarbonyl carbamate derivatives of mutilin have potentantimicrobial activity.

Accordingly the present invention provides a compound of formula (IA) or(IB):

in which:

-   R¹ is optionally substituted C₍₁₋₆₎alkyl or C₍₃₋₆₎cycloalkyl or    optionally substituted heterocyclyl;-   R² is vinyl or ethyl;-   R³ is H, OH or F, and R⁴ is H, or R³ is H and R⁴ is F, and R⁵ and R⁶    together form an oxo group; or-   R³ and R⁴ is each H, R⁵ is OH or H and R⁶ is H, or R⁵ is H and R⁶ is    OH or H.

Examples of compounds of formula (IA) include those in which R³ and R⁴are both hydrogen, and R⁵ and R⁶ together form an oxo group.

Further examples of compounds of formula (IA) include those in which R³is OH and R⁴ is hydrogen, and R⁵ and R⁶ together form an oxo group.

Representative substituents for R¹ include 1, 2 or 3 substituentsselected from hydroxy, C₍₁₋₆₎alkylsulfonyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclyl,carbamoyl, C₍₁₋₆₎alkylcarbonylamino, C₍₁₋₆₎alkylsulfonylamino, anduriedo.

Preferred substituents for R¹ include hydroxy, fluoro,carbamoylC₍₁₋₆₎alkyl, carbamoyloxy, carbamoyloxyC₍₁₋₆₎alkyl, cyano,cyanoC₍₁₆₎alkyl, ureido (optionally substituted by C₍₁₋₆₎alkyl),guanidino, C₍₁₋₆₎alkylguanidino, amidino, C₍₁₋₆₎alkylamidino, oxamoyl,amino (optionally substituted by, for example, 1 or 2 substituents whichmay be the same or different selected from oxamoyl (optionallysubstituted on N by hydroxy(C₁₋₆)alkyl), acyl, (C₁₋₆)alkylsulphonyl,(C₁₋₆)alkyl (optionally substituted by, for example, hydroxy), acyl,arylcarbonyl and carbamoyl(C₁₋₆)alkyl), mono- anddi-(C₁₋₆)alkylamino(C₁₋₆)alkyl, aminoC(₁₋₆)alkyl,amino(C₁₋₆)alkylcarbonyl, sulphonylamino, aminosulphonyl and R⁷R⁸NCOwherein R⁷ and R⁸ which may be the same or different is each selectedfrom hydrogen, (C₁₋₆)alkyl, (C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl,cyano(C₁₋₆)alkyl and heteroaryl(C₁₋₆)alkyl or R⁷ and R⁸ together withthe nitrogen atom to which they are attached form a heterocyclyl ring.

Representative examples of R¹ include:

-   (a) C₍₁₋₆₎alkyl optionally substituted by 1, 2 or 3 substituents    selected from

heteroaryl optionally substituted by 1, 2 or 3 substituents selectedfrom C₍₁₋₆₎alkyl, hydroxyC₍₁₋₆₎alkyl, amino, aminoC₍₁₋₆₎alkyl,carbamoyl, cyano and hydroxy,

heterocyclyl optionally substituted by 1, 2 or 3 substituents selectedfrom oxo, C₍₁₋₆₎alkyl, hydroxyC₍₁₋₆₎alkyl, aminosulphonyl, carbamoyl andacyl,

amino substituted by 1 or 2 substituents which may be the same ordifferent selected from oxamoyl (optionally substituted on N byhydroxy(C₁₋₆)alkyl), acyl, (C₁₋₆)alkylsulphonyl, (C₁₋₆)alkyl (optionallysubstituted by, for example, hydroxy), and carbamoyl(C₁₋₆)alkyl,

carbamoyloxy, ureido substituted by C₍₁₋₆₎alkyl, C₍₁₋₆₎alkylsulphonyl,hydroxy, halogen, C₍₁₋₆₎alkylthio, and R⁷R⁸NCO wherein R⁷ and R⁸ whichmay be the same or different is each selected from hydrogen,(C₁₋₆)alkyl, (C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl, cyano(C₁₋₆)alkyl andheteroaryl(C₁₋₆)alkyl or R⁷ and R⁸ together with the nitrogen atom towhich they are attached form a heterocyclyl ring,

-   (b) C₍₃₋₆₎cycloalkyl substituted by amino, carbamoyl or    di-C₍₁₋₆₎alkylaminoC₍₁₋₆₎alkyl, and-   (c) a 4- to 6-membered heterocyclyl ring or two fused 5-membered    heterocyclyl rings containing 1 or 2 heteroatoms in each ring (for    example tetrahydrofuran, 1,3-dioxane, hexahydrofuro[3,2-b]furan,    trimethylenesulphide, tetrahydrothiophene, azetidine, pyrrolidine,    isoxazolidine or piperidine) optionally substituted by 1, 2 or 3    substituents selected from C₍₁₋₆₎alkyl, hydroxy, hydroxyC₍₁₋₆₎alkyl,    C₍₁₋₆₎alkylsulphonyl, C₍₁₋₆₎alkoxy, oxo, oxamoyl, carbamoyl,    carbamoylC(₁₋₆₎alkyl, carbamoyloxy, carbamoyloxyC₍₁₋₆₎alkyl,    aminoC₍₁₋₆₎alkylcarbonyl, amino (optionally substituted by oxamoyl),    C₍₁₋₆₎alkoxyimino, azido, cyano, cyano(C₁₋₆)alkyl, and    heteroarylcarbonyl (optionally substituted by, for example, amino).

Typical examples of R¹ include, for example, methyl, hydroxyethyl, andmethylsulfonylethyl.

Preferred examples of R¹ include C₍₁₋₆₎alkyl substituted bycarbamoyloxy, C₍₁₋₆₎alkyl substituted by hydroxy anddi-N—C₍₁₋₆₎alkylamino, C₍₃₋₆₎cycloalkyl substituted bydi-C₍₁₋₆₎alklaminoC₍₁₋₆₎alkyl and a 4- or 5-membered heterocyclyl ringcontaining one nitrogen atom optionally substituted by oxo.

Representative examples of groups for R³ include H and OH.

Representative examples of groups for R⁴ include H.

Preferably, R³ and R⁴ is each H.

Preferably, R⁵ and R⁶ together form an oxo group.

When used herein, the term “aryl” refers to, unless otherwise defined,phenyl or naphthyl. A substituted aryl group comprises up to five,preferably up to three substituents.

Suitable substituents for an aryl group, including phenyl when formingpart of a benzyl group, include, for example, and unless otherwisedefined, halogen, (C₁₋₆)alkyl, aryl, aryl(C₁₋₆)alkyl, (C₁₋₆)alkoxy,(C₁₋₆)alkoxy(C₁₋₆)alkyl, halo(C₁₋₆)alkyl, aryl(C₁₋₆)alkoxy, hydroxy,nitro, cyano, azido, amino, mono- and di-N-(C₁₋₆)alkylamino, acylamino,arylcarbonylamino, acyloxy, carboxy, carboxy salts, carboxy esters,carbamoyl, mono- and di-N-(C₁₋₆)alkylcarbamoyl, (C₁₋₆)alkoxycarbonyl,aryloxycarbonyl, ureido, guanidino, (C₁₋₆)alkylguanidino, amidino,(C₁₋₆)alkylamidino, sulphonylamino, aminosulphonyl, (C₁₋₆)alkylthio,(C₁₋₁₆)alkylsulphinyl, (C₁₋₁₆)alkylsulphonyl, heterocyclyl, heteroaryl,heterocyclyl(C₁₋₆)alkyl and heteroaryl(C₁₋₆)alkyl. In addition, twoadjacent ring carbon atoms may be linked by a (C₃₋₅)alkylene chain, toform a carbocyclic ring.

Further suitable substituents for an aryl group include substituted(C₁₋₆)alkyl, substituted amino, amino(C₁₋₆)alkyl,amino(C₁₋₆)alkylcarbonyl, oxamoyl, carbamoyl(C₁₋₆)alkyl,cyano(C₁₋₆)alkyl, acyl, carbamoyloxy, carbamoyloxy(C₁₋₆)alkyl andoptionally substituted heteroarylcarbonyl.

When used herein, the terms “alkyl” and “alkenyl” refer to (individuallyor as part of alkoxy or alkenyloxy) straight and branched groupscontaining up to six carbon atoms.

When used herein, the terms “cycloalkyl” and “cycloalkenyl” refer togroups having from three to eight ring carbon atoms.

When substituted, an alkyl, alkenyl, cycloalkyl or cycloalkenyl groupmay comprise up to four substituents, preferably up to two substituents.

Suitable substituents for alkyl, alkenyl, cycloalkyl or cycloalkenylgroups include aryl, heteroaryl, heterocyclyl, (C₁₋₆)alkoxy,(C₁₋₆)alkylthio, aryl(C₁₋₆)alkoxy, aryl(C₁₋₆)alkylthio, amino, mono- ordi-(C₁₋₆)alkylamino, cycloalkyl, cycloalkenyl, carboxy and estersthereof, carbamoyl, ureido, guanidino, (C₁₋₆)alkylguanidino, amidino,(C₁₋₆)alkylamidino, (C₁₋₆)acyloxy, azido, hydroxy, and halogen.

Further suitable substituents for alkyl, alkenyl, cycloalkyl orcycloalkenyl groups include substituted aryl, substituted heteroaryl,substituted heterocyclyl, substituted amino, carbamoyloxy, ureidosubstituted by (C₁₋₆)alkyl, (C₁₋₆)alkylsulphonyl, mono- ordi-(C₁₋₆)alkylamino(C₁₋₆)alkyl, and carbamoyl substituted by 1 or 2substituents which may be the same or different selected from(C₁₋₆)alkyl, (C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl, cyano(C₁₋₆)alkyl andheteroaryl(C₁₋₆)alkyl, or carbamoyl substituted by 2 substituents whichtogether with the nitrogen atom to which they are attached form aheterocyclyl ring.

Preferred substituents for cycloalkyl include hydroxy, amino, carbamoyland di-C(₁₋₆)alkylaminoC(₁₋₆)alkyl.

When used herein the terms “heterocyclyl” and “heterocyclic” refer to,unless otherwise defined, non-aromatic, single, bridged and fused, ringssuitably containing up to four heteroatoms in each ring, each of whichis selected from oxygen, nitrogen and sulphur. Each heterocyclic ringpreferably has from 4 to 7, preferably 5 or 6, ring atoms. A fusedheterocyclic ring system may include carbocyclic rings and need includeonly one heterocyclic ring.

Representative examples of heterocyclyl rings include non-aromatic,saturated rings containing 1 or 2 heteroatoms and having 4 to 7, inparticular 5 or 6, atoms in each ring, for example tetrahydrofuran,1,3-dioxolane, 1,3-dioxane, hexahydrofuro[3,2-b]furan,trimethylenesulphide, tetrahydrothiophene, azetidine, pyrrolidine,oxazolidine, isoxazolidine, piperidine, piperazine, morpholine andthiomorpholine. Preferably the heterocyclyl ring contains 1 or 2nitrogen atoms.

When substituted, a heterocyclyl group may comprise up to threesubstituents. Preferably a substituent for a heterocyclyl group isselected from oxo, C(₁₋₆)alkylimino, and the groups hereinbefore definedas suitable aryl substituents.

Representative examples of non-aromatic, unsaturated heterocyclyl ringssubsituted by oxo include, for example, pyridone and optionallysubstituted uracil.

When used herein, the term “heteroaryl” suitably includes, unlessotherwise defined, a mono- or bicyclic heteroaromatic ring systemcomprising up to four, preferably 1 or 2, heteroatoms each selected fromoxygen, nitrogen and sulphur. Each ring may have from 4 to 7, preferably5 or 6, ring atoms. A bicyclic heteroaromatic ring system may include acarbocyclic ring.

Representative examples of heteroaryl rings includes pyrazole,isoxazole, pyridine, pyrimidine and pyrazine.

When substituted, a heteroaryl group may comprise up to threesubstituents. Preferably a substituent for a heteroaryl group isselected from the group hereinbefore defined as suitable arylsubstituents.

When used herein, the term “acyl” includes formyl and(C₁₋₆)alkylcarbonyl.

When used herein, the term “sulfonyl” includes (C₁₋₆)alkylsulfonyl.

The term halo or halogen includes fluoro, chloro, bromo and iodo.

When used herein, the term “substituted amino”, unless otherwisedefined, refers to an amino group substituted by 1 or 2 substituents.

Suitable substituents for an amino group include oxamoyl (optionallysubstituted on N by hydroxy(C₁₋₆)alkyl), acyl, (C₁₋₆)alkylsulphonyl,(C₁₋₆)alkyl (optionally substituted by, for example, hydroxy), andcarbamoyl(C₁₋₆)alkyl.

It will be appreciated that depending on the substituents at the C-14position certain compounds of the present invention may comprise one ormore chiral centres so that compounds may exist as stereoisomers,including diastereomers and epimers. The present invention covers allsuch stereoisomers, and mixtures thereof, including racemates.

Depending on the substituents, two or more diastereoisomers may bepossible. In that situation the present invention includes theindividual diastereoisomers and mixtures thereof

The 2-hydroxy-substituted compounds of formula (I) are of the 2 (S)configuration.

Preferred compounds of the invention include:

-   Mutilin 14-[N-(2-carbamoyloxyethoxycarbonyl)]carbamate;-   Mutilin 14-[N-(2-oxopyrrolidin-3-(S)-yloxycarbonyl)]carbamate;-   Mutilin 14-[N-(azetidinyloxycarbonyl)]carbamate hydrochloride;-   Mutilin 14-[N-(pyrrolidin-3-(R)-yloxycarbonyl)]carbamate;-   Mutilin 14-[N-(pyrrolidin-3-(S)-yloxycarbonyl)]carbamate;-   Mutilin    14-[N-(3-dimethylamino-2-hydroxyprop-1-yloxycarbonyl)]carbamate; and-   Mutilin    14-[N-(1-(dimethylaminomethyl)cyclopropoxycarbonyl)]carbamate.

Particularly preferred compounds of the invention include:

-   Mutilin 14-[-(pyrrolidin-3-(R)-yloxycarbonyl)]carbamate; and-   Mutilin 14-[N-(pyrrolidin-3-(S)-yloxycarbonyl)]carbamate.

The compounds of this invention may be in crystalline or non-crystallineform, and, if crystalline, may optionally be hydrated or solvated. Thisinvention includes within its scope stoichiometric hydrates as well ascompounds containing variable amounts of water.

The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitable atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight.

Compounds of the invention that contain a basic group such as an aminosubstituent may be in the form of a free base or an acid addition salt.Compounds having an acidic group such as a carboxy substituent may be inthe form of a pharmaceutically acceptable salt. Compounds of theinvention having both a basic and an acidic centre may be in the form ofzwitterions, acid addition salt of the basic centre or alkali metalsalts (of the carboxy group). Pharmaceutically acceptable salts arepreferred.

Pharmaceutically acceptable acid-addition salts include those describedby Berge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19.Suitable salts include the hydrochloride, maleate, and methanesulfonate;particularly the hydrochloride.

Pharmaceutically acceptable salts for acidic groups include thosedescribed by Berge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66,1-19. Suitable salts include alkali metal salts such as the sodium andpotassium salts.

Compounds of the present invention may be readily prepared from a (epi-)mutilin or a 19,20-dihydro-mutilin compound by adapting procedures wellknown in the art for forming carbamate groups. Representative processesare described in WO 97/25309 and WO 98/05659 (SmithKline Beecham plc).In a preferred process, a chloroformate derivative is reacted withsilver cyanate followed by reaction with an alcohol. It will beappreciated that the (epi-) mutilin or a 19,20-dihydro-mutilin compoundmay provide either the alcohol or the chloroformate component.

Accordingly, the present invention provides a process for preparing acompound of formula (IA) or (IB) which process comprises reacting achloroformate compound of formula (IIA) or (IIB):

in which:

-   P is hydrogen or an hydroxy-protecting group;-   R^(2A), R^(3A), R^(4A), R^(5A) and R^(6A) are R², R³, R⁴, R⁵ and R⁶    as defined for formulae (IA) and (IB) or a group convertible to R²,    R³, R⁴, R⁵ and R⁶ respectively;-   with silver cyanate, in a suitable aprotic solvent, for instance    dichloromethane, followed by treating with an alcohol compound of    formula (III):    R^(1A)OH  (III)-   in which R^(1A) is R¹ as defined for formulae (IA) and (IB) or a    group convertible to R¹;-   in the presence of pyridine, in a carbamate forming reaction, and    thereafter, and if so needed;-   converting P to hydrogen, and, if necessary, converting an R^(2A),    R^(3A), R^(4A), R^(5A) and R^(6A) group to an R², R³, R⁴, R⁵ and R⁶    group.

It will be appreciated that the process may be reversed so that achloroformate derivative of the alcohol of formula (III) is reacted withsilver cyanate, followed by addition of a pleuromutilin derivative offormula (IVA) or (IVB):

in which P, R^(2A), R^(3A), R^(4A), R^(5A) and R^(6A) are ashereinbefore defined.

Preparation of the chloroformate compounds of formula (IIA) and (IIB)are described in WO 97/25309 and WO 98/05659 (SmithKline Beecham plc).

Conversion of an R^(1A), R^(2A), R^(3A), R^(4A), R^(5A) or R⁶A group toan R¹, R², R³, R⁴, R⁵ and R⁶ group typically arises if a protectinggroup is needed during the above reactions or during the preparation ofthe reactants by the procedures described below.

When P is a hydroxyl protecting group, a preferred protecting group isacyl, for example so that —OP is trifluoroacetoxy or dichloroacetoxy.When the intended R³, R⁵ or R⁶ is also hydroxyl, then R^(3A), R^(5A) andR^(6A) is also preferably acyloxy, for example acetoxy ordichloroacetoxy. Hydroxyl groups at positions 11, 3 and 2 (as groups OP,R^(5A) and R^(6A) and R^(3A)) may be protected using, for example,trifluoroacetic anhydride or dichloroacetic anhydride and pyridine intetrahydrofuran or N-trifluoroacetyl-imidazole in tetrahydrofuran at 0°C. After the reaction described above with (III) is complete, theprotecting acyl groups may be removed to restore the hydroxyl groups,for instance by hydrolysis e.g. using NaOH in either MeOH ortetrahydrofuran/water solution, or sodium hydrogen carbonate in aqueousethanol.

Suitable hydroxy, carboxy and amino protecting groups are those wellknown in the art and which may be removed under conventional conditionsand without disrupting the remainder of the molecule. A comprehensivediscussion of the ways in which hydroxy, carboxy and amino groups may beprotected and methods for cleaving the resulting protected derivativesis given in for example Protective Groups in Organic Chemistry, T. W.Greene and P. G. M. Wuts, (Wiley-Interscience, New York, 2nd edition,1991). Particularly suitable hydroxy protecting groups include, forexample, triorganosilyl groups such as, for instance, trialkylsilyl andalso organocarbonyl and organooxycarbonyl groups such as, for instance,acetyl, allyloxycarbonyl and 4-methoxybenzyloxycarbonyl. Particularlysuitable carboxy protecting groups include alkyl and aryl esters, forinstance methyl, ethyl and phenyl. Particularly suitable aminoprotecting groups include alkoxycarbonyl groups such astert-butoxycarbonyl and benzyloxycarbonyl.

R^(2A) is typically the R² group vinyl, and this may be converted to thealternative R² ethyl group by hydrogenating the vinyl group to form anethyl group, typically by hydrogenation over a palladium catalyst (e.g.10% palladium-on-carbon) in a solvent such as ethyl acetate, ethanol,dioxane, or tetrahydrofuran.

R^(3A) is typically hydrogen, fluoro or protected hydroxyl, such asacyloxy. After the coupling reaction, if required, protecting acylgroups may be removed to restore the hydroxyl groups by hydrolysis e.g.using NaOH in MeOH, or sodium hydrogen carbonate in aqueous ethanol.

A compound of formula (IA) in which R³ and R⁴ are both hydrogen and R⁵and R⁶ form an oxo group may also be prepared from an epi-mutilinstarting material. Accordingly, in a further aspect, the presentinvention provides a process for preparing a compound of formula (IA) inwhich R³ and R⁴ are both hydrogen and R⁵ and R⁶ form an oxo group, whichcomprises reacting a chloroformate epi-mutilin compound of formula(IIC):

wherein R^(2A) is as hereinbefore defined;

-   with silver cyanate and then a compound of formula (III), as    hereinbefore defined; under carbamate forming conditions as    hereinbefore described;-   and then treating the product with an acid;-   and where required or desired converting an R^(1A) group to an R¹    group and an R^(2A) group to an R² group.

Alternatively, a chloroformate derivative of the alcohol of formula(III) may be treated initially with silver cyanate, followed bytreatment with an epi-mutilin compound of formula (IID):

under carbamate forming conditions; and then

-   treating the product with an acid,-   and where required or desired converting an R^(1A) group to an R¹    group and an R^(2A) group to an R² group.

The acid treatment indicated above converts the epi-mutilinconfiguration to the usual mutilin nucleus of formula (IA). Typicallythis conversion is carried out by treatment with conc. HCl or Lukasreagent (conc. HCl saturated with ZnCl₂) in dioxane.

It should be appreciated that it may be necessary to interconvert oneR¹, R², R³, R⁴, R⁵ or R⁶ group to another R¹, R², R³, R⁴, R⁵ or R⁶group. This typically arises when one compound of formula (IA/B) is usedas the immediate precursor of another compound of formula (IA/B) or whenit is easier to introduce a more complex or reactive substituent at theend of a synthetic sequence. A substituent group in R¹ can be convertedinto another substituent group using one of the general methods forfunctional group transformation described in the literature (e.g. acarboxylic ester can be hydrolysed to a carboxylic acid with base; anacid can be converted into an amide; a tert-butoxycarbonylamino groupcan be converted into an amine by treatment with trifluoroacetic acid;an amino group can be alkylated or acylated), provided that the methodchosen is compatible with other functional groups in the molecule (e.g.the ketone at C-3 in the pleuromutilin nucleus).

Functional group transformations are well known in the art and aredescribed in, for instance, Comprehensive Organic Functional GroupTransformations, eds. A. R. Katritzky, O. Meth-Cohn, and C. W. Rees(Elsevier Science Ltd., Oxford, 1995), Comprehensive Organic Chemistry,eds. D. Barton and W. D. Ollis (Pergamon Press, Oxford, 1979), andComprehensive Organic Transformations, R. C. Larock (VCH PublishersInc., New York, 1989).

Compounds of formulae (IIA) in which R^(3A) and R^(4A) are hydrogen,(IIB) and (IIC) may be readily prepared according to methods describedin the literature, for example G. Schulz and H. Berner, Tetrahedron,1984, 40, 905, and in WO 97/25309 and WO 98/05659 (SmithKline Beecham).Where necessary, and as hereinbefore described, saponification of theC-14 ester may be carried out at an appropriate stage.

Compounds of formula (IIA) in which R^(3A) is hydroxyl or fluoro may beprepared from pleuromutilin, via an intermediate 2-diazo compound, thepreparation of which is described by G. Schulz and H. Berner inTetrahedron, 1984, 40, 905. Where necessary, saponification of the C-14ester group may be carried out at an appropriate stage usingconventional techniques such as sodium hydroxide or sodium methoxide inmethanol or aqueous tetrahydrofuran solution.

The intermediate 2-diazo compound may be reacted with a carboxylic acidto give a 2-acyloxy-mutilin derivative. Suitably, reaction withdichloroacetic acid gives a 2-dichloroacetoxy-mutilin derivative, whichcan be deprotected as described above to provide the (2S)-2-hydroxyderivative, at an appropriate stage.

Compounds of formula (IIA) in which R^(3A) is fluoro may be obtained byreacting 2-diazo-mutilin with a source of hydrogen fluoride.Conveniently, the hydrogen fluoride source is an amine complex ofhydrogen fluoride such as hydrogen fluoride-pyridine. The reaction maybe carried out in an anhydrous solvent (e.g. diethyl ether,tetrahydrofuran, 1,2-dimethoxyethane), at a temperature of −15° C. to25° C. This reaction produces (2S)-2-fluoro derivatives.(2R)-2-Fluoro-mutilin derivatives may be prepared by treating the(2S)-isomer with a base (e.g. sodium hydroxide or potassium hydroxide inethanol). This will usually produce a mixture of (2S) and (2R)-isomersthat may be separated using conventional techniques such aschromatography and crystallisation.

Compounds of formula (IIA) in which R⁵ is hydroxy and R⁶ is hydrogen maybe prepared according to methods described in the literature, forexample, by reduction of a mutilin with lithium aluminium hydride asdescribed by Birch et al, Tetrahedron, 1966, supp 8 (II), 359-387; or byreduction with lithium tri-tert-butoxyaluminohydride in dioxane asdescribed by G. Schultz et al, Tetrahedron, 1984, 40, 905-917.

Compounds of formula (IIA) in which R⁵ is hydrogen and R⁶ is hydroxy maybe prepared according to the methods described in the literature, forexample, by reduction of a mutilin with lithium and methanol in liquidammonia, as described by Birch et al, Tetrahedron, 1966, supp 8 (II),359-387.

Compounds of formula (IIA) in which R⁵ and R⁶ are both hydrogen may beprepared according to the method of G. Schultz et al, Tetrahedron, 1984,40, 905-917, by reduction with potassium hydroxide and hydrazine inrefluxing diethylene glycol.

The compounds of the present invention may contain a chiral centre, andtherefore the above processes may produce a mixture of diastereoisomers.A single diastereoisomer may be prepared by separating such a mixture ofdiastereoisomers by conventional techniques such as chromatography orfractional crystallisation.

The compounds of this invention may be in crystalline or non-crystallineform, and, if crystalline, may optionally be hydrated or solvated. Whensome of the compounds of this invention are allowed to crystallise orare recrystallised from organic solvents, solvent of crystallisation maybe present in the crystalline product. Similarly, some of the compoundsof this invention may be crystallised or recrystallised from solventscontaining water. In such cases water of hydration may be present in thecrystalline product. Crystallisation procedures will usually producestoichiometric hydrates. Compounds containing variable amounts of watermay be produced by processes such as lyophilisation.

The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitable atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight. An impure orless pure form of a compound according to the invention may, forexample, be used in the preparation of a more pure form of the samecompound or of a related compound (for example a correspondingderivative) suitable for pharmaceutical use.

The present invention also includes pharmaceutically acceptable saltsand derivatives of the compounds of the invention. Salt formation may bepossible when one of the substituents carries an acidic or basic group.Salts may be prepared by salt exchange in conventional manner.

Acid-addition salts may be pharmaceutically acceptable ornon-pharmaceutically acceptable. In the latter case, such salts may beuseful for isolation and purification of the compound of the invention,or intermediates thereto, and will subsequently be converted into apharmaceutically acceptable salt or the free base.

The compounds of the present invention and their pharmaceuticallyacceptable salts or derivatives have antimicrobial properties and aretherefore of use in therapy, in particular for treating microbialinfections in animals, especially mammals, including humans, inparticular humans and domesticated animals (including farm animals). Thecompounds may be used for the treatment of infections caused by, forexample, Gram-positive and Gram-negative bacteria and mycoplasmas,including, for example, Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecalis, Streptococcus pyogenes,Streptococcius agalactiae, Streptococcus pneumoniae, Haemophilus sp.,Neisseria sp., Legionella sp., Chlamydia sp., Moraxella catarrhalis,Mycoplasma pneumoniae, and Mycoplasnia gallisepticum.

In addition, compounds of this invention are active against bacterialorganisms which are resistant (including multiply-resistant) to otheranti-bacterial agents, for instance, β-lactam antibiotics such as, forexample, methicillin; macrolides; and quinolones. Such bacterialorganisms include, for example, methicillin resistant Staphylococciusaureus (MRSA) and drug-resistant Streptococcus pneumoniae (DRSP).Compounds of the present invention are therefore useful in the treatmentof infections caused by these bacteria.

The present invention also provides a method of treating microbialinfections in animals, especially in humans and in domesticated mammals,which comprises administering a compound of the invention or apharmaceutically acceptable salt or derivative or solvate thereof, or acomposition according to the invention, to a patient in need thereof.

Compounds of the present invention show good activity against Chlamydiapneumoniae. This has been implicated in heart disease, in particular inpromoting vascular infection (see for instance FR 2,771,008-A1, HoechstMarion Roussel S A). Accordingly, in a further aspect, the presentinvention provides a method of preventing C pneumoniae-inducedatherosclerosis which method comprises treating a subject in needthereof with an effective amount of a compound of formula (I). Acompound of formula (I) may also be used in combination with ananti-atherosclerotic agent, to reduce the incidence of heart attack andother cardiac events. Representative examples of anti-atheroscleroticagents include the class of cholesterol-lowering compounds referred togenerically as “statins”, for instance atorvastatin (Lipitor, WarnerLambert), pravastatin (Pravachol), simvastatin (Lipovas, Merck) andcerivastatin (Baycol, Bayer). It has also been suggested that Chlamydiapneumoniae may contribute to Alzheimer's Disease. Accordingly, in afurther aspect, the present invention provides a method of treatingAlzheimer's Disease which method comprises treating a subject in needthereof with an effective amount of a compound of formula (I).

The invention further provides the use of a compound of the invention ora pharmaceutically acceptable salt or derivative or solvate thereof inthe preparation of a medicament for use in the treatment of microbialinfections.

Compounds of the present invention may be used to treat skin and softtissue infections and acne, by topical application. Accordingly, in afurther aspect the present invention provides the use of a compound ofthe invention or a pharmaceutically acceptable salt or derivative orsolvate thereof in the preparation of a medicament adapted for topicaladministration for use in the treatment of skin and soft tissueinfections and also in the treatment of acne in humans.

Compounds of the present invention may be also used for the eliminationor reduction of nasal carriage of pathogenic bacteria such as S. aureus,H. influenzae, S. pneumonia and M. catarrhalis, in particularcolonisation of the nasospharynx by such organisms, by theadministration of a compound of the present invention thereto.Accordingly, in a further aspect, the present invention provides for theuse of a compound of the invention or a pharmaceutically acceptable saltor derivative or solvate thereof in the manufacture of a medicamentadapted for administration to the nasal cavity, for reducing oreliminating the nasal carriage of pathogenic organisms. Preferably, themedicament is adapted for focussed delivery to the nasopharynx, inparticular the anterior nasopharynx.

Such reduction or elimination of nasal carriage is believed to be usefulin prophylaxis of recurrent acute bacterial sinusitis or recurrentotitis media in humans, in particular in reducing the number of episodesexperienced by a patient over a given period of time or increasing thetime intervals between episodes. Accordingly, in a further aspect, thepresent invention provides for the use of a compound of the invention ora pharmaceutically acceptable salt or derivative or solvate thereof inthe manufacture of a medicament adapted for administration to the nasalcavity, for prophylaxis of recurrent acute bacterial sinusitis orrecurrent otitis media

Compounds of the present invention are also useful in treating chronicsinusitis. Accordingly, in a further aspect, the present inventionprovides for the use of a compound of the invention or apharmaceutically acceptable salt or derivative or solvate thereof in themanufacture of a medicament, for treating of chronic sinusitis.

The compounds according to the invention may suitably be administered tothe patient at a daily dosage of from 1.0 to 50 mg/kg of body weight.For an adult human (of approximately 70 kg body weight), from 50 to 3000mg, for example about 1500 mg, of a compound according to the inventionmay be administered daily. Suitably, the dosage for adult humans is from5 to 20 mg/kg per day. Higher or lower dosages may, however, be used inaccordance with normal clinical practice.

To lessen the risk of encouraging the development of resistant organismsduring prophylaxis of recurrent otitis media or recurrent acutebacterial sinusitis, it is preferred to administer the drug on anintermittent, rather than a continual, basis. In a suitable intermittenttreatment regimen for prophylaxis of recurrent otitis media or recurrentsinusitis, drug substance is administered on a daily basis, for a smallnumber of days, for instance from 2 to 10, suitably 3 to 8, moresuitably about 5 days, the administration then being repeated after aninterval, for instance, on a monthly basis over a period of months, forinstance up to six months. Less preferably, the drug substance may beadministered on a continuing, daily basis, over a prolonged period, forinstance several months. Suitably, for prophylaxis of recurrent otitismedia or recurrent acute bacterial sinusitis (RABS), drug substance isadministered once or twice a day. Suitably, drug substance isadministered during the winter months when bacterial infections such asrecurrent otitis media and recurrent sinusitis tend to be moreprevalent. The drug substance may be administered at a dosage of from0.05 to 1.00 mg, typically about 0.1 to 0.2 mg, in each nostril, once ortwice a day.

More generally, the compounds and compositions according to theinvention may be formulated for administration in any convenient way foruse in human or veterinary medicine, by analogy with other antibiotics.

Accordingly, in a further aspect, the present invention provides apharmaceutical composition comprising a compound of the invention or apharmaceutically acceptable salt or derivative or solvate thereoftogether with a pharmaceutically acceptable carrier or excipient.

The compounds and compositions according to the invention may beformulated for administration by any route, for example oral, topical orparenteral. The compositions may, for example, be made up in the form oftablets, capsules, powders, granules, lozenges, creams, syrups, spraysor liquid preparations, for example solutions or suspensions, which maybe formulated for oral use or in sterile form for parenteraladministration by injection or infusion.

Tablets and capsules for oral administration may be in unit dosage form,and may contain conventional excipients including, for example, bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricants, forexample magnesium stearate, talc, polyethylene glycol or silica;disintegrants, for example potato starch; and pharmaceuticallyacceptable wetting agents, for example sodium lauryl sulphate. Thetablets may be coated according to methods well known in normalpharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives, including, for example, suspending agents, forexample sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel orhydrogenated edible fats; emulsifying agents, for example lecithin,sorbitan monooleate or acacia; non-aqueous vehicles (which may includeedible oils), for example almond oil, oily esters (for exampleglycerine), propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid; and, ifdesired, conventional flavouring and colour agents.

Compositions according to the invention intended for topicaladministration may, for example, be in the form of ointments, creams,lotions, eye ointments, eye drops, ear drops, nose drops, nasal sprays,impregnated dressings, and aerosols, and may contain appropriateconventional additives, including, for example, preservatives, solventsto assist drug penetration, and emollients in ointments and creams. Suchtopical formulations may also contain compatible conventional carriers,for example cream or ointment bases, ethanol or oleyl alcohol forlotions and aqueous bases for sprays. Such carriers may constitute fromabout 1% to about 98% by weight of the formulation; more usually theywill constitute up to about 80% by weight of the formulation.

Compositions according to the invention intended for topicaladministration, in addition to the above, may also contain a steroidalanti-inflammatory agent; for example, betamethasone.

Compositions according to the invention may be formulated assuppositories, which may contain conventional suppository bases, forexample cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteraladministration may conveniently be in fluid unit dosage forms, which maybe prepared utilizing the compound and a sterile vehicle, water beingpreferred. The compound, depending on the vehicle and concentrationused, may be either suspended or dissolved in the vehicle. In preparingsolutions, the compound may be dissolved in water for injection andfilter-sterilised before being filled into a suitable vial or ampoule,which is then sealed. Advantageously, conventional additives including,for example, local anaesthetics, preservatives, and buffering agents canbe dissolved in the vehicle. In order to enhance the stability of thesolution, the composition may be frozen after being filled into thevial, and the water removed under vacuum; the resulting dry lyophilisedpowder may then be sealed in the vial and a accompanying vial of waterfor injection may be supplied to reconstitute the liquid prior to use.Parenteral suspensions may be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterisisation cannot be accomplished by filtration. Thecompound may instead be sterilised by exposure to ethylene oxide beforebeing suspended in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in such suspensions in order to facilitateuniform distribution of the compound.

A compound or composition according to the invention is suitablyadministered to the patient in an anti-microbially effective amount.

A composition according to the invention may suitably contain from0.001% by weight, preferably (for other than spray compositions) from 10to 60% by weight, of a compound according to the invention (based on thetotal weight of the composition), depending on the method ofadministration.

When the compositions according to the invention are presented in unitdosage form, for instance as a tablet, each unit dose may suitablycomprise from 25 to 1000 mg, preferable from 50 to 500 mg, of a compoundaccording to the invention.

Representative compositions of the present invention include thoseadapted for intranasal administration, in particular, those that willreach into the nasopharynx. Such compositions are preferably adapted forfocussed delivery to, and residence within, the nasopharynx. The term‘focussed delivery’ is used to mean that the composition is delivered tothe nasopharynx, rather than remaining within the nares. The term‘residence’ within the nasopharynx is used to mean that the composition,once delivered to the nasopharynx, remains within the nasopharynx over acourse of several hours, rather than being washed away more or lessimmediately. Preferred compositions include spray compositions andcreams. Representative aqueous sprays are described in WO 99/21855(SmithKline Beecham). Representative oily spray and cream compositionsare described in WO 98/14189 (SmithKline Beecham).

Suitably, the drug substance is present in compositions for nasaldelivery in between 0.001 and 5%, preferably 0.005 and 3%, by weight ofthe composition. Suitable amounts include 0.5% and 1% by weight of thecomposition (for oily compositions and creams) and from 0.01 to 0.2%(aqueous compositions).

Spray compositions according to the present invention may be deliveredto the nasal cavity by spray devices well known in the art for nasalsprays, for instance an air lift pump. Preferred devices include thosethat are metered to provide a unit volume of composition, preferablyabout 100 μl, and optionally adapted for nasal administration byaddition of a modified nozzle.

The invention is illustrated by the following Examples.

EXAMPLES Intermediate 1

To bis-2-hydroxyethylamine (0.63 g) in THF (30 ml) was addedpolydimethylaminomethylpolystyrene (3.2 mmol/g, 2.5 g) andmethanesulfonyl chloride (0.358 ml), and the mixture stirred for 20 h.The solid was filtered and extracted with THF (4 ml). The combined THFsolutions were passed through a 1 g SCX cartridge and evaporated to acolourless gum (0.395 g). NMR analysis indicated a 19:3:10 ratio ofN-monosulfonyl:N,O-disulfonyl:N,O,O-trisulfonyl product, which was usedwithout further purification in general procedure A.

Intermediate 2

To 3-hydroxypropylamine (0.45 g) in THF (30 ml) was addedpolydimethylaminomethylpolystyrene (3.2 mmol/g, 2.5 g) andmethanesulfonyl chloride (0.358 ml), and the mixture stirred for 20 h.The solid was filtered and extracted with ThF (4 ml). The combined THFsolutions were passed through a 1 g SCX cartridge and evaporated to acolourless gum (0.271 g). NMR analysis indicated a 27:73 ratio ofN-monosulfonyl:N,O-disulfonyl product, which was used without furtherpurification in general procedure A.

Intermediate 3 N-(2-hydroxyethyl)oxalamide

Ethyl oxamate (1.17 g, 10 mmol) in methanol (15 ml) was added toaminoethanol (0.732 g, 12 mmol) and the mixture stirred for 3 h. Theprecipitated product was filtered off, washed with methanol, and driedin vacuo to give the title compound as a white powder (0.94 g, 71%); NMR[(CD₃)₂SO] 3.18 (2H, q, J=6 Hz), 3.43 (2H, q, J=6 Hz), 4.72 (1H, t, J=6Hz), 7.75 (1H, br s), 8.03 (1H, br s), and 8.43 (1H, bt).

Intermediate 4 N-(2-Hydroxy-1,1-dimethylethyl)oxalamide

Prepared as in Intermediate 3, using 2-amino-2-methylpropanol. Theproduct did not precipitate out, even on adding diethyl ether. Thesolution was passed through a SCX cartridge, to remove excess amine,then evaporated to give the title compound as a white powder (1.54 g,96%); NMR [(CD₃)₂SO] 1.25 (6H, s), 3.36 (2H,d), 5.07 (1H, t), 7.74 (1H,s), 7.78 (1H, br s), and 8.04 (1H, br s).

Intermediate 5 N-((3S,4R)-4-Hydroxytetrahydrofuran-3-yl)oxalamide

Prepared as in Intermediate 3, using (3R,4S)-4-aminotetrahydrofuran-3-olhydrochloride [Schaus et al, J. Org. Chem., 62, 4197 (1997)] andtriethylamine, to give the title compound as a pale brown powder (1.13g, 65%); NMR [(CD₃)₂SO] 3.45-3.56 (2H, m), 3.84-3.94 (2H, m), 3.98-4.01(1H, m), 4.19-4.24 (1H, m), 5.24 (1H, t), 7.79 (1H, br s), 8.06 (1H, brs), and 8.66 (1H, d).

Intermediate 6 N-(3-hydroxypropyl)oxalamide

Prepared as in Intermediate 3, using 3-aminopropanol to give the titlecompound as a white powder (1.15 g, 75%); NMR [(CD₃)₂SO] 1.6 (2H, m),3.18 (2H, m), 3.42 (2H, m), 4.47 (1H, t), 7.73 (1H, br s), 8.0 (1H, brs), and 8.63 (1H, t).

Intermediate 7 6-Hydroxymethyl-1H-pyridin-2-one

1H-Pyridin-2-one-6-carboxylic acid (2.51 g, 18 mmol) was suspended indry THF (30 ml) and 1,2-dimethoxyethane (10 ml) under argon. Lithiumaluminium hydride (1.0 M in diethyl ether) (30 ml, 30 mmol) was addedand the mixture heated to reflux. After 67 h heating was stopped, themixture cooled to 0° C. and acetic acid (7 ml) added. The solvent wasremoved in vacuo and the residue purified by flash column chromatographyon silica gel, eluting with 20-30% methanol in dichloromethane, to givethe title compound as tan needles (0.714 g, 32%); δ_(H) (400 MHz; CD₃OD)4.47 (2H, s), 6.38 (1H, d, J 6.8), 6.41 (1H, d, J 9.2), 7.56 (1H, dd, J6.8, 9.2); m/z J 6.8, 9.2); m/z (ES+) 126 (100%, [MH]⁺).

Intermediate 82,6-Di(bis(tert-butoxycarbonyl)amino)-4-hydroxymethylpyrimidine

(a) Methyl 2,6-diaminopyrimidine-4-carboxylate2,6-Diaminopyrimidine-4-carboxylic acid [Nishigaki et al, Chem. Pharm.Bull., 18, 1003 (1970)](3.27 g, 21 mmol) was suspended in methanol (200ml) which had been saturated with hydrogen chloride. The mixture wasrefluxed for 5 h then evaporated to dryness to give the crude titlecompound (3.53 g, 100%).

(b) Methyl 2,6-di(bis(tert-butoxycarbonyl)amino)pyrimidine-4-carboxylateCrude methyl 2,6-diaminopyrimidine-4-carboxylate (2.52 g, 15 mmol) wassuspended in dry tetrahydrofuran (200 ml) then treated sequentially withdi-tert-butyl dicarbonate (19.6 g, 90 mmol), triethylamine (16.7 ml, 120mmol), and 4 dimethylaminopyridine (0.092 g, 0.75 mmol). This mixturewas refluxed gently for 16 h, evaporated, dissolved in diethyl ether,washed with water and brine, dried and evaporated to give a dark redgum. This residue was purified by flash chromatography, eluting with0-18% ethyl acetate in petroleum ether, to give the title compound as anoff-white foam (4.58 g, 54%); m/z (APCI+) 569 [MH]⁺100%.

(c) 2,6-Di(bis(tert-butoxycarbonyl)amino)-4-hydroxymethylpyrimidineMethyl 2,6-di(bis(tert-butoxycarbonyl)amino)pyrimidine-4-carboxylate(0.284 g, 0.5 mmol) was dissolved in tetrahydrofuran (4.5 ml) and water(0.5 ml) then sodium borohydride (0.038 g, 1 mmol) added. The mixturewas stirred for 0.5 h, then diluted with ethyl acetate, washed withdilute sodium chloride solution and brine, dried and evaporated to givethe crude product as a white foam. This was purified by flashchromatography, eluting with 15-30% ethyl acetate in petroleum ether, togive the title compound as a white foam (0.183 g, 68%); LC/MS (APCI+)541 [MH]⁺100%.

Intermediate 9 5-Hydroxymethylpyrazine-2-carboxylic Acid Amide

Pyrazinecarboxylic acid amide (0.492 g, 4 mmol) was suspended inmethanol (8 ml), water (4 ml) and concentrated sulphuric acid (0.22 ml,4 mmol), then iron (II) sulphate heptahydrate (0.334 g, 1.2 mmol) wasadded and the mixture degassed. To this was then addedhydroxylamine-O-sulphonic acid (1.36 g, 12 mmol). After 6 h moresulphuric acid and iron (II) sulphate heptahydrate were added. After afurther 1 h the mixture was adjusted to pH 7, evaporated, and theresidue extracted with methanol. This solution was pre-absorbed ontosilica then purified by chromatography, eluting with 0-10% ‘2 M ammoniain methanol’ in dichloromethane, to give the title compound as anoff-white solid (0.102 g, 17%); NMR [(CD₃)₂SO] 4.72 (2H, d), 5.72 (1H,t), 7.79 (1H, br s), 8.22 (1H, br s), 8.73 (1H, d), and 9.09 (1H, d).

Intermediate 10 4-Amino-5-cyano-2-hydroxymethylpyrimidine

To hydroxyacetamidine hydrochloride (3.3 g) in ethanol (40 ml) was addedethanolic sodium ethoxide prepared from ethanol (25 ml) and sodium (0.76g). The sodium chloride was filtered off and the solution added toethoxymethylene malondinitrile (3.66 g) in ethanol (30 ml). After 1 hthe precipitated orange title product was removed by filtration (2.27 g,45%); NMR [(CD₃)₂SO] 4.4 (d, J=6.2 Hz, 2H), 5.1 (t, J=6.2 Hz, 1H), 7.9(bs, 1H), 8.6 (s, 1H).

Intermediate 11 4-Amino-5-cyano-2-hydroxymethyl-6-methylpyrimidine

To hydroxyacetamidine hydrochloride (3.3 g) in ethanol (40 ml) was addedethanolic sodium ethoxide prepared from ethanol (25 ml) and sodium (0.76g). The sodium chloride was filtered off and the solution added to1-ethoxyethylene malondinitrile (4.08 g) in ethanol (30 ml). After 1 hthe precipitated yellow title product was removed by filtration (4.29 g,75%); NMR [(CD₃)₂SO] 2.4 (3H, s), 4.3 (d, J=6.1 Hz, 2H), 5.0 (t, J=6.1Hz, 1H), 7.7 (bs, 1H). 7.7 (bs, 1H).

Intermediate 12 4-Amino-5-(N-Boc-aminomethyl)-2-hydroxymethylpyrimidine

To the compound of Intermediate 10 (1.5 g) in THF (180 ml) under argonwas added 1 M lithium aluminium hydride in THF (20 ml) and the reactionstirred at 20° C. for 24 h. Water (1.5 ml) in THF (10 ml) was addeddropwise and the precipitated solid filtered off and extracted withethanol. The combined solutions were evaporated to dryness under reducedpressure and the residue taken up in water (50 ml) and treated withdi-t-butyl dicarbonate (5 g) in THF (50 ml). The mixture was stirred for24 h, then extracted with ethyl acetate. The extract was washed withbrine, dried and evaporated to a yellow gum which was purified bychromatography on silica eluting with ethyl acetate to give the titleproduct as a yellow gum (0.41 g, 26%); NMR (CDCl₃) 1.45 (s, 9H), 4.2 (d,J=6.8 Hz, 2H), 4.55 (s, 2H), 4.95 (bt, 1H), 6.1 (bs, 2H), 8.0 (s, 1H).

Intermediate 13 3-Hydroxy-3-methylpyrrolidine-1-carboxylic AcidTert-butyl Ester

3-Oxo-pyrrolidine-1-carboxylic acid tert-butyl ester [Hofmann, J. Agric.Food Chem., 46, 3902 (1998)] (0.322 g, 1.74 mmol) was dissolved indiethyl ether (5 ml), cooled with an ice bath, and treated withmethylmagnesium bromide (3M in ether, 0.64 ml, 1.91 mmol). The reactionwas stirred for 1 h then more methylmagnesium bromide added, and themixture stirred for 63 h. The reaction was quenched with aqueousammonium chloride and extracted with diethyl ether (x3). The combinedorganic extracts were dried and evaporated. The residue was purified byflash chromatography, eluting with 0-50% ethyl acetate in petroleumether, to give the title compound as a sticky off-white solid (0.129 g,37%); NMR (CDCl₃) 1.41 (3H, s), 1.46 (9H, s), 1.61 (1H, s), 1.79-1.92(2H, m), and 3.17-3.58 (4H, m).

Intermediate 14 N-Boc-(2S,4R)-4-Hydroxypyrrolidine-2-carboxamide

To Boc-hydroxyproline dicyclohexylamine salt (8.25 g) in acetonitile (25ml) was added di-t-butyl dicarbonate (6 g), pyridine (1 ml) and ammoniumbicarbonate (2 g). After stirring at 20° C. for 24 h, the mixture wasdiluted with water (50 ml) and extracted twice with ethyl acetate(100+50 ml). The organic extracts were combined, dried and evaporated togive a colourless foam (6.1 g), which was shown to be highly impure. Athird extraction of the aq layer with ethyl acetate (100 ml) gave thetitle product as a white foam, (1.5 g). NMR [(CD₃)₂SO] major rotamer:1.34, (s, 9H), 1.8 (m, 1H), 2.0 (m, 1H), 3.23 (m, 1H), 3.37(m, 1H), 4.08(m, 1H), 4.21 (m, 1H), 4.95 (d, 3.2 Hz, 1H), 6.88 (bs, 1H), 7.33 (bs,1H); minor rotamer: 1.39 (s, 9H), 1.8 (m, 1H), 2.0 (m, 1H), 3.23 (m,1H), 3.37(m, 1H), 4.08 (m, 1H), 4.21 (m, 1H), 4.95 (d, 3.2 Hz, 1H), 6.81(bs, 1H), 7.3 (bs, 1H).

Intermediate 15 N-Boc-(2S,3S)-3-Hydroxypyrrolidine-2-carboxamide

To N-Boc-(2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (1.36 g) in DMF(6 ml) added ammonium bicarbonate (0.5 g) and dicyclohexylcarbodiimide(1.3 g). After stirring at 20° C. for 20 h the reaction was concentratedby evaporation under reduced pressure, and the residue triturated withethyl acetate. The soluble material was purified by chromatography onsilica eluting with 50-100% (5% acetic acid in diethyl ether) in hexaneto give the title material as a colourless gum, (0.5 g). NMR (CD₃OD)major rotamer: 1.43, (s, 9H), 1.88 (m, 1H), 2.0 (m, 1H), 3.55 (m, 2H),4.03(s, 1H), 4.31 (bs, 1H); minor rotamer: 1.47, (s, 9H), 1.7 (m, 1H),2.0 (m, 1H), 3.55 (m, 2H), 4.09(s, 1H), 4.34 (d, J=4 Hz, 1H).

Intermediate 16N-Boc-(2S,4R)-4-Hydroxy-2-t-butyldimethylsilyloxymethylpyrrolidine

To N-Boc-(2S,4R)-4-hydroxy-2-hydroxymethylpyrrolidine (0.199 g, 0.92mmol) in DMF (2 ml) was added imidazole (0.125 g) andt-butyldimethylsilyl chloride (0.14 g). After 1.5 h, the reaction wasconcentrated by evaporation under reduced pressure, and the residuedissolved in diethyl ether, washed with water and brine, dried andevaporated. Purification by chromatography on silica gel eluting with0-30% ethyl acetate in hexane gave the title material, (0.15 g, 49%).NMR (CDCl₃) 0.02 (s, 3H), 0.03 (s, 3H), 0.9 (s, 9H), 1.45 (s, 9H), 1.9(bm, 9H), 2.2 (bm, 1H), 3.3-4.0 (bm, 5H), 4.5 (m, 1H).

Intermediate 17 N-Boc-(2S,4R)-2-Carbamoylmethyl-4-hydroxypyrrolidine

To N-Boc-(2S,4R)-4-hydroxy-2-hydroxymethylpyrrolidine (0.105 g in THF (5ml) cooled to 0° C. under argon was added carbonyl diimidazole (121 mg).After 3 h aqueous ammonia (20 M, 0.25 ml) was added and the reactionstirred for 17 h. The reaction mixture was evaporated to dryness underreduced pressure and the residue purified by chromatography on silicaeluting with 80-100% ethyl acetate in hexane to give the title productas a colourless gum (45 mg, 31%); NMR (CDCl₃) 1.47 (s, 9H), 2.05 (m,3H), 3.4-3.6 (m, 2H), 4.154.3 (m, 3H), 4.45 (m, 1H), 4.75 (bs, 2H).

Intermediate 18 N-Boc-Isoxazolidin-4-ol

Isoxazolidin-4-ol hydrochloride (0.1 g) in dichloromethane (2 ml) wastreated with triethylamine (0.14 ml) and di-t-butyl dicarbonate (0.22g). The reaction was stirred at 20° C. for 18 h. The reaction mixturewas washed with water, 5% aqueous citric acid and brine, dried andevaporated to a colourless gum. Purification by chromatography on silicagel eluting with 0-100% ethyl acetate in hexane gave the title material,(0.15 g, 49%). NMR (CDCl₃) 1.5 (s, 9H), 3.68 (dd, J=2 Hz, 12 Hz, 1H),3.73 (dd, J=5 Hz, 12 Hz, 1H), 3.94 (d, J=3 Hz, 2H), 4.76 (m, 1H).

Intermediate 19 (3R,3aR,6S,6aR)-6-azido-hexahydrofuro[3,2-b]furan-3-ol

(a) (3R,3aS,6R,6aR)-6-Hydroxy-hexahydro-furo[3,2-b]furan3-O-methanesulfonate (3R,3a,6R,6aR)-Hexahydro-furo[3,2-b]furan-3,6-diol(3.21 g, 22 mmol) was dissolved in dry dichloromethane (100 ml), cooledin an ice/salt bath and treated sequentially with triethylamine (3.35ml, 24 mmol) and methanesulfonyl chloride (1.55 ml, 20 mmol). Themixture was stirred for 20 h while warming to room temperature. Thesolution was washed with water then brine, dried and evaporated to givea brown oil (2.9 g). This residue was purified by flash chromatography,eluting with 0-8% methanol in dichloromethane, to give the titlecompound as an off-white gum (1.35 g, 35%).

(b) (3R,3aR,6S,6aR)-6-azido-hexahydrofuro[3,2-b]furan-3-ol(3R,3aS,6R,6Hydroxy-hexahydro-furan[3,2-b]furan 3-O-methanesulfonate(0.768 g, 4 mmol) was dissolved in dry dimethylformamide (8 ml) thensodium azide (0.325 g, 5 mmol) added. The mixture was heated at 55° C.for 20 h, 75° C. for 2 h, 110° C. for 24 h and 120° C. for 48 h. Waterwas then added and Water was then added and the mixture extracted withethyl acetate. The combined organic extracts were dried and evaporatedto give a pale orange solid (0.5 g). This residue was purified by flashchromatography, eluting with 30-70% ethyl acetate in petroleum ether, togive the title compound as a colourless solid (0.346 g, 51%); NMR(CDCl₃) 2.55 (1H, d), 3.60 (1H, dd), 3.87 (1H, dd), 3.97 (1H, dd), 4.08(2H, m), 4.32 (1H, m), 4.47 (1H, d), and 4.63 (1H, m).

Intermediate 20 (S)-3-Hydroxy-4-methoxyimino-pyrrolidine-1-carboxylicacid tert-butyl Ester

(a) 3a,7a-dihydroxy-octahydro-4,8-dioxa-2,6-diaza-s-indacene-2,6-dicarboxylicacid di-tert-butyl ester Dimethyl sulfoxide (279 μl, 3.94 mmol) wasadded dropwise to a stirred solution of oxalyl chloride (258 μl, 2.95mmol) in THF (10 ml) at −78° C. After 10 min(3S,4S)-3,4-dihydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester(0.5 g, 2.46 mmol) [Nagel, Angew. Chem., 96, 425 (1984)] in THF (5 ml)was added. The mixture was stirred at −78° C. for 95 min, after whichtriethylamine (1.7 ml, 12.3 mmol) was added. Stirring was continued fora further 20 min, after which the mixture was allowed to warm to roomtemperature. Citric acid (5% solution) (20 ml) was added and the mixtureextracted with ethyl acetate. The organic extracts were combined, dried(MgSO₄), filtered, and concentrated in vacuo then purified by flashcolumn chromatography on silica gel, eluting with 50-70% ethyl acetatein petroleum ether to give 3a,7a-dihydroxy-octahydro-4,8-dioxa-2,6-diaza-s-indacene-2,6-dicarboxylicacid di-tert-butyl ester (195 mg, 20%); m/z (ES+) 425 (100%, [MNa]⁺).

(b) (S)-3-Hydroxy-4-methoxyimino-pyrrolidine-1-carboxylic acidtert-butyl ester To 3a,7a-dihydroxy-octahydro-4,8-dioxa-2,6-diaza-s-indacene-2,6-dicarboxylicacid di-tert-butyl ester (188 mg, 0.47 mmol) in DCM (10 ml) was addedmethoxylamine hydrochloride (94 mg, 1.12 mmol), followed bytriethylamine (157 μl, 1.12 mmol). After stirring at room temperaturefor 44 hours the mixture was concentrated in vacuo then purified byflash column chromatrography on silica gel, eluting with 20-30% ethylacetate in petroleum ether to give the title compound as a white solid(156 mg, 73%); δ_(H) (400 MHz; CDCl₃) 1.47 (9H, s), 2.87 (1H, d),3.43-3.50 (1H, m), 3.72-3.83 (1H, br m), 3.92 (3H, s), 4.12 (2H, AB q),4.70-4.75 (1H, m); m/z (ES+) 253 (87%, [MNa]⁺), 157 (100%).

Intermediate 21 1-Hydroxycyclopropanecarboxamide

To 1-hydroxycyclopropanecarboxylic acid (0.20 g, 1.96 mmol) in dry DMF(15 ml) was added 1-hydroxy-7-azabenzotriazole (0.27 g, 2.15 mmol).Ammonia (0.037 g, 2.15 mmol) was added, followed by1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.38 g,2.15 mmol) after 30 min. The mixture was stirred under argon for 68 h,water (1 ml) was added, and the mixture concentrated in vacuo. Theresidue was taken up in sodium hydrogen carbonate solution (1 ml),filtered through hydromatrix gel and washed through with ethyl acetate.The filtrate was concentrated in vacuo then purified by flash columnchromatography on silica gel, eluting with ethyl acetate to give thetitle compound as a colourless solid (0.048 g, 24%); δ_(H) (400 MHz;CD₃OD) 0.94-0.98 (2H, m), 1.18-1.21 (2H, m).

Intermediate 22 1-Dimethylaminomethylcyclopropanol Hydrochloride

(a) 1-Hydroxycyclopropanecarboxylic acid dimethylamide To1-hydroxycyclopropanecarboxylic acid (1.20 g, 1.8 mmol) in dry DMF (15ml) was added 1-hydroxy-7-azabenzotriazole (1.76 g, 12.9 mmol).Dimethylamine in THF (2.0 M; 6.5 ml, 12.9 mmol) was added, followed by1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (2.48 g,12.9 mmol) after 30 min. The mixture was stirred under argon for 22 h,water (2 ml) was added, and the mixture concentrated in vacuo. Theresidue was taken up in sodium hydrogen carbonate solution and extractedwith ethyl acetate. The organic extracts were combined, dried (MgSO₄),filtered, and concentrated in vacuo then purified by flash columnchromatography on silica gel, eluting with 90-100% ethyl acetate inpetroleum ether to give 1-hydroxycyclopropanecarboxylic aciddimethylamide as a colourless solid (1.15 g, 76%); δ_(H) (400 MHz;CDCl₃) 0.91-0.94 (2H, m), 1.03-1.06 (2H, m), 3.11 (6H, br s), 4.53 (1H,br s); m/z (ES+) 130 (100%, [MH]⁺).

(b) 1-(Dimethylaminomethyl)cyclopropanol hydrochloride A solution of1-hydroxycyclopropanecarboxylic acid dimethylamide (435 mg, 3.4 mmol) indiethyl ether (15 ml) was cooled in an ice bath. Lithium aluminiumhydride (1.0 M in diethyl ether; 8.4 ml, 8.4 mmol) was added and themixture heated to reflux. After 3 h sodium hydroxide solution (40%; 2ml) was added to the ice-cooled mixture. The precipitate which formedwas filtered off and washed with ethyl acetate. The filtrate was dried(MgSO₄), filtered, acidified with methanolic hydrogen chloride, thenconcentrated in vacuo to give the title compound as a colourless solid(466 mg, 91%); δ_(H) (400 MHz; CD₃OD) 0.73-0.76 (2H, m), 0.90-0.93 (2H,m), 2.96 (6H, s), 3.26 (2H, s); m/z (ES+) 116 (100%, [MH]⁺).

Intermediates 23 and 24

Intermediates 23 and 24 were prepared by general procedure A. The crudereaction was suspended in dilute sodium hydrogen carbonate solution andextracted with ethyl acetate. The aqueous was acidified withhydrochloric acid (2 M) and extracted with ethyl acetate. The combinedorganic extracts were dried (MgSO₄), filtered, and concentrated invacuo.

Int. R Alcohol MW Yield LC/MS 23

Ref A, * 465 72% 488 (ES⁺) [MNa]⁺ 20% 24

C, * 493 67% 516 (ES⁺) [MNa]⁺ 35% * Starting alcohol is t-butyl ester.

References

Reference A: Juraj and Cushman, Tetrahedron, 48, 8601 (1992).

Intermediate 25 (3R,4S)-4-Boc-amino-3-hydroxytetrahydrofuran

(3R,4S)-4-Amino-3-hydroxytetrahydrofuran hydrochloride (Scott et al, J.Org. Chem., 62, 4197 (1997); 0.75 g, 5.37 mmol) in dichloromethane (10ml) was treated with triethylamine (0.8 ml) and t-butyl dicarbonate (1.3g). The reaction was stirred at 20° C. for 18 h. The reaction mixturewas diluted with ether (20 ml) washed with water, 5% aqueous citric acidand brine, dried and partially evaporated under reduced pressure, togive a white solid. This was collected, washed with hexane and driedunder reduced pressure to give the title compound, (0.64 g, 58%). NMR(CDCl₃) 1.45 (9H, s), 3.0 (1H, bs), 3.6 (1H, dd), 3.7 (1H, dd), 3.95(1H, m), 4.1 (2H, m), 4.28 (1H, m), 4.7 (1H, bm).

Example 1 Mutilin 14-[N-(2-methylsulfonylethoxycarbonyl)]carbamate

(a) (3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin14-[N-(2-methylsulfonylethoxycarbonyl)]carbamate 2-(Methylsulfonyl)ethylchloroformate (Wolters et al., Synthesis 563, 1996; 0.41 g, 2.2 mmol)was dissolved in dry dichloromethane (10 ml) then silver cyanate (0.495g, 3.3 mmol) added. After stirring for a few minutes(3R)-3-deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin (0.668 g, 2 mmol)was added, and stirring continued for 2.5 h. The mixture was thenfiltered through kieselguhr and evaporated. The residue was purified byflash chromatography, eluting with 0-50% ethyl acetate in petroleumether, to give (3R)-3-deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin14-[N-(2-methylsulfonylethoxycarbonyl)]carbamate (0.78 g, 74%); LC/MS(APCI−) 526 (100%, [M−H]⁻).

(b) Mutilin 14-[N-(2-methylsulfonylethoxycarbonyl)]carbamate.(3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin14-[N-(2-methylsulfonylethoxycarbonyl)]carbamate (0.468 g, 0.89 mmol)was dissolved in 1,4-dioxane (12 ml) and cooled to 5-10° C. Lukasreagent (3 ml) was then added and the mixture stirred for 4.5 h withcooling and for 3 h at room temperature. The mixture was diluted withethyl acetate, washed with water (x2), saturated sodium hydrogencarbonate, and brine, dried and evaporated. The residue was purified byflash chromatography, eluting with 40-80% ethyl acetate in petroleumether, to give the title compound as a white foam (0.362 g, 79%); LC/MS(APCI−) 512 (100%, [M−H]⁻).

General Procedure A, for the Preparation of Mutilin14-[N-(alkoxycarbonyl)]carbamates

(3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin 14-chloroformate (WO97/25309; 0.16 g, 0.4 mmol) was dissolved in dry dichloromethane (2.5ml) and added to silver cyanate (0.09 g, 0.6 mmol). To this stirredsuspension was added pyridine (0.008 g, 0.1 mmol) in dry dichloromethane(0.1 ml). After 1.5 min the appropriate alcohol (ca 1 equiv. formono-alcohols or 2-7.5 equiv. for diols) in dry dichloromethane or DMFwas added. The mixture was stirred for 3.5 h then filtered throughkieselguhr and evaporated. The residue was dissolved in 1,4-dioxane (4ml), concentrated hydrochloric acid (2 ml) added, and the mixturestirred for 6 h. After evaporation the residue was suspended in dilutesodium hydrogen carbonate solution and extracted with ethyl acetate(x2). The combined organic extracts were washed with brine, dried andevaporated. The residue was purified by column chromatography on silicagel, eluting with 20-100% ethyl acetate in petroleum ether then 3-4%methanol in ethyl acetate, to give the required product.

Examples 2-6

Examples 2-6 were prepared by general procedure A.

LC/MS (APCI−) Example R¹ MW Yield [M − H]⁻ 2 Me 421 21% 420, 100% 3HO(CH₂)₂— 451 31% 450, 100% 4

528 11% 527, 100% 5

478 29% 477, 100% 6

492 71% 491, 100%

Examples 7-68

Examples 7-68 were prepared by general procedure A. For basic productsthe aqueous work-up was omitted and the residue was purified by columnchromatography on silica gel eluting with 0-15% 2M methanolic ammonia indichloromethane. The alcohol starting material was commerciallyavailable unless otherwise indicated.

Example R Alcohol MW Yield LC/MS 7

C 464 56% 463 (APCI−) [M − H]⁻ 100% 8

Ref 1 528 30% 527 (APCI−) [M − H]⁻ 100% 9

Int. 1 572 13% 571 (APCI−) [M − H]⁻ 100% 10

Int. 2 542 17% 565 (APCI+) [MNa]⁺ 100% 11

Int. 3 521 46% 520 (APCI−) [M − H]⁻ 100% 12

Int. 4 549 13% 572 (ES+) (MNa+) 100% 13

Int. 5 563  9% 586 (ES+) (MNa+) 100% 14

C 565 33% 588 (ES+) (MNa+) 100% 15

Int. 6 535 40% 534 (APCI−) [M − H]⁻ 100% 16

Ref 2 494 59% 493 (APCI−) [M − H]⁻ 100% 17

Ref 3 508  7% 507 (APCI−) [M − H]⁻ 100% 18

Ref 3 522 48% 521 (APCI−) [M − H]⁻ 50% 19

Ref 3 522 45% 521 (APCI−) [M − H]⁻ 50% 20

C 481 47% 480 (APCI−) [M − H]⁻ 100% 21

Ref 4 501 59% 524 (ES+) (MNa+) 100% 22

C 478 31% 477 (APCI−) [M − H]⁻ 100% 23

C 525 10% 524 (APCI−) [M − H]⁻ 100% 24

C 490 25% 489 (APCI−) [M − H]⁻ 100% 25

C 490  9% 489 (APCI−) [M − H]⁻ 100% 26

Ref 5 490 26% 513 (ES+) [MNa]⁺ 100% 27

C 504 55% 503 (APCI−) [M − H]⁻ 100% 28

C 504 42% 503 (APCI−) [M − H]⁻ 100% 29

Ref 6 506 44% 505 (APCI−) [M − H]⁻ 100% 30

Ref 7 506 43% 505 (APCI−) [M − H]⁻ 100% 31

C 502 55% 501 (APCI−) [M − H]⁻ 100% 32

C 498 31% 497 (APCI−) [M − H]⁻ 100% 33

Int. 7 514 13% 515 (ES+) [MH]⁺ 100% 34

Int. 8 529 68% 530 (ES+) (MH+) 100% 35

Int. 9 542 20% 541 (APCI−) [M − H]⁻ 40% 36

Ref 8 542 11% 541 (APCI−) [M − H]⁻ 60% 37

Int. 10 539 44% 603 (ES+) [MNa.MeCN]^(+ 100%) 38

Int. 11 553 43% 617 (ES+) [MNa.MeCN]^(+ 100%) 39

Int. 12 543 21% 544 (ES+) [MH]⁺ 100% 40

C 531 14% 530 (APCI−) [M − H]⁻ 100% 41

C 559 25% 558 (APCI−) [M − H]⁻ 100% 42

C 542 37% 541 (APCI−) [M − H]⁻ 100% 43

Ref 9, # 462 66% 463 (ES+) [MH]⁺ 100% 44

Ref 10, # 476 29% 475 (ES−) [M − H]⁻ 100% 45

Ref 10, # 476 16% 475 (ES−) [M − H]⁻ 100% 46

C, # 490 53 489 (APCI−) [M − H]⁻ 100% 47

Ref 9, # 490 70% 491 (ES+) [MH]⁺ 100% 48

Int. 13, # 490 67% 489 (APCI−) [M − H]⁻ 100% 49

Ref 11, # 490 34% 491 (ES+) [MN]⁺ 100% 50

Ref 12, # 506 58% 507 (ES+) [MH]⁺ 100% 51

Ref 13, # 506 62% 505 (APCI−) [M − H]⁻ 100% 52

Int. 14, # 519  2% 518 (ES−) [M − H]⁻ 100% 53

Int. 15, # 519  5% 518 (ES−) [M − H]⁻ 100% 54

Int. 16, # 506 26% 505 (ES−) [M − H]⁻ 100% 55

Int. 17, # 549 28% 548 (ES−) [M − H]⁻ 100% 56

Int. 18, # 478 34% 501 (ES+) [MNa]⁺ 100% 57

Ref 14, # 506 82% 507 (ES+) [MH]⁺ 100% 58

C 478 66% 479 (ES+) (MH⁺) 100% 59

C 508 11% 507 (ES−) [M − H]⁻ 100% 60

C 538  7% 539 (ES−) [M − H]⁻ 100% 61

C 492 62% 491 (APCI−) [M − H]⁻ 100% 62

C, @ 508  3% 507 (ES−) [M − H]⁻ 100% 63

C, @ 508 30% 507 (ES−) [M − H]⁻ 100% 64

C 550 14% 549 (ES−) [M − H]⁻ 100% 65

C, # 519 79% 518 (APCI−) [M − H]⁻ 100% 66

C 520 24% 519 (ES−) [M − H]⁻ 100% 67

C 568 20% 591 (ES+) [MNa]⁺ 100% 68

C 561  2% 560 (ES−) [M − H]⁻ 100% C commercially available alcohol; #Starting alcohol is N-Boc derivative; @ both examples isolated from thereaction of the diol. References: 1. Japan Kokai, JP50149622 (1975). 2.Gormley et al, J. Org. Chem., 45, 1450 (1980). 3. Najer et al, Bull.Soc. Chim. Fr., 1142 (1954). 4. Starrett et al. J. Med. Chem., 37, 1857(1994). 5. Bentley et al, J. Chem. Soc. Chem. Commun., 231 (1995). 6.Nordmark-Werke GmbH, DE2538424 (1975). 7. Seneci et al, J. Chem. Soc.Perkin Trans. I, 2345 (1994). 8. Van Dobeneck et al, Chem. Ber., 105,3611 (1972). 9. Lee et al, Bioorg. Med. Chem. Lett., 10, 1063 (2000).10. Kucznierz et al, J. Med. Chem., 41, 4983 (1998). 11. Cesare et al,J. Med. Chem., 35, 4205 (1992). 12. Okada et al, Chem. Pharm. Bull., 41,132 (1993). 13. Heffner et al, J. Amer. Chem. Soc., 114, 10181 (1992).14. WO 00/09491, PCT/US/18377 (2000).

NMR data for Example 16: δ_(H) (CDCl₃) 0.77 (3H, d), 0.88 (3H, d),1.0-1.8 (15H, m), 2.05-2.35 (5H, m), 3.35 (1H, dd), 4.2-4.4 (4H, m),4.68 (2H, br s), 5.22 (1H, dd), 5.37 (1H, dd), 5.75 (1H, dd), and 7.08(1H, s).

NMR data for Example 26: δ_(H) (400 MHz; CD₃OD) 0.76 (3H, d), 0.93 (3H,d), 1.1-1.2 (1H, br m), 1.16 (3H, s), 1.3-1.7 (6H, m), 1.45 (3H, s), 1.8(1H, m), 2.1-2.4 (6H, s), 2.6 (1H, m), 3.4 (2H, m) 3.5 (1H, d), 5.15-5.2(2H, m), 5.3(1H, t) 5.71 (1H, d), 6.34 (1H, dd).

NMR data for Example 43: δ_(H) (400 MHz; CD₃OD) 0.74 (3H, d), 0.93 (3H,d), 1.10-1.18 (1H, br m), 1.16 (3H, s), 1.33-1.50 (3H, m), 1.45 (3H, s),1.53-1.72 (3H, m), 1.78-1.86 (1H, m), 2.10-2.38 (5H, m), 3.50 (1H, d),4.154.20 (2H, m), 4.39-4.46 (2H, m), 5.16 (1H, d), 5.19 (1H, s),5.29-5.34 (1H, m), 5.71 (1H, d), 6.34 (1H, dd).

NMR data for Example 44: δ_(H) (400 MHz; CDCl₃) 0.77 (3H, d), 0.88 (3H,d), 1.1 (1H, m), 1.18 (3H, s), 1.45 (3H, s) 1.3-2.4 (15H, m), 3.0-3.25(4H, m), 3.35 (1H, d), 5.2 (1H, d), 5.2 (1H, m)5.38 (1H, bd), 5.74 (1H,d) and 6.52 (1H, dd). (NH resonances not seen)

NMR data for Example 45: δ_(H) (400 MHz; CDCl₃) 0.77 (3H, d), 0.88 (3H,d), 1.1 (1H, m), 1.18 (3H, s), 1.45 (3H, s) 1.3-2.4 (15H, m), 2.9-3.1(4H, m), 3.35 (1H, d), 5.2 (1H, d), 5.3 m)5.37 (1H, d), 5.74 (1H, d),6.52 (1H, dd) 7.4 (1H, b).

NMR data for Example 63: δ_(H) (400 MHz; CDCl₃) 0.76 (3H, d), 0.88 (3H,d), 1.1 (1H, d), 1.18 (3H, s), 1.45 (3H, s) 1.3-2.4 (16H, m), 2.3 (6H,s) 3.35 (1H, d) 3.9 (1H, m), 4.05 (1H, m), 4.25 (1H, m), 5.22 (1H, d),5.36 (1H, 4)), 5.74 (1H, d), 6.52 (1H, dd) 7.1 (1H, b).

General Procedure B, for the Preparation of Mutilin14-[N-(alkoxycarbonyl)]carbamates

(a) 11-Trifluoroacetylmutilin 14-chloroformate To11-trifluoroacetylmutilin (WO 97/25309) (4.16 g, 10 mmol) in dry THF (30ml) at 0° C. under argon was added pyridine (0.808 ml, 10 mmol) andtriphosgene (1.09 g, 3.67 mmol, portionwise). After stirring for 10 minat 0° C., hexane (50 ml) was added and the mixture washed with water andbrine, and dried. After evaporation the chloroformate was obtained as awhite solid (4.3 g, 90%).

(b)) Mutilin 14-[N-(alkoxycarbonyl)]carbamates 11-Trifluoroacetylmutilin14-chloroformate (0.191 g, 0.4 mmol) was dissolved in drydichloromethane (2.5 ml) and added to silver cyanate (0.09 g, 0.6 mmol).To this stirred suspension was added pyridine (0.008 g, 0.1 mmol) in drydichloromethane (0.1 ml). After 1.5 min the appropriate alcohol (0.4-2mmol) in dry dichloromethane or DMF was added. The mixture was stirredfor 3.5 h, then treated with water (0.1 ml), and ethyl acetate added (10ml). This mixture was then filtered through hydromatrix gel andkieselguhr. After evaporation the residue was dissolved in ethanol (8ml) and saturated aqueous sodium hydrogen carbonate (8 ml) added. Themixture was stirred for 6 h, then filtered and the filtrate extractedwith ethyl acetate. The filtered solid was slurried in the aqueous phaseand extracted with ethyl acetate. The combined organic extracts werewashed with brine, dried and evaporated. The residue was purified bycolumn chromatography on silica gel, eluting with 20-100% ethyl acetatein petroleum ether then 0-10% methanol in dichloromethane, to give therequired product.

Examples 69-90

Examples 69-90 were prepared by general procedure B. In the case ofExample 77 the crude material (after having been filtered throughhydromatrix gel and kieselguhr and then evaporated) was treated withtrifluoroacetic acid (1 ml) for 1 h then concentrated in vacuo. Afterevaporation the residue was taken up in ethanol (10 ml) and saturatedsodium hydrogen carbonate (10 ml). The mixture was stirred for 2.5 h,then worked up as in the general procedure.

Example R Alcohol MW Yield LC/MS 69

C 509  15% 508 (ES−) [M − H]⁻ 100% 70

C{circumflex over ( )} 493  27% 492 (APCI−) [M − H]⁻ 100% 71

C{circumflex over ( )} 493  10% 492 (APCI−) [M − H]⁻ 100% 72

C 535   9% 534 (APCI−) [M − H]⁻ 100% 73

C, $ 535   5% 558 (ES+) [MNa]⁺ 50% 74

C, $ 535  50% 558 (ES+) [MNa]⁺ 100% 75

Int. 19 560  81% 559 (APCI−) [M − H]⁻ 100% 76

Ref 15 533  27% 534 (ES+) [MH]⁺ 100% 77

Int. 20 519  31% 520 (ES+) [MH]⁺ 100% 78

C 492  43% 493 (ES+) [MH]⁺ 100% 79

Ref 15 521  25% 522 (ES+) [MH]⁺ 100% 80

Ref 16 507  42% 530 (ES+) [MNa]⁺ 100% 81

C 563  13% 562 (ES−) [M − H]⁻ 100% 82

Int. 21 490  43% 513 (ES+) [MNa]⁺ 100% 83

Int. 22 504  16% 505 (ES+) [MH]⁺ 100% 84

C 504  34% 503 (ES−) [M − H]⁻ 100% 85

C 504  28% 503 (APCI−) [M − H]⁻ 100% 86

Ref 17 518  59% 517 (ES−) [M − H]⁻ 100% 87

Ref 18 518  10% 517 (APCI−) [M − H]⁻ 100% 88

C 532  33% 531 (APCI−) [M − H]⁻ 100% 89

Ref 19 467 8.5% 466 (APCI−) [M − H]⁻ 100% 90

Ref 20 511   6% 510 (ES−) [M − H]⁻ 100% C commercially availablealcohol; {circumflex over ( )}both examples isolated from reaction withthe commercially available mixture of 1,3-dioxan-5-ol and1,3-dioxolane-4-methanol; $ both isomers obtained from the reaction withthe same alcohol. References: 15. Kamiya et al, EP 264091 (1988). 16.Miyahara et al, Chem. Pharm. Bull., 33, 497 (1985) 17. Kopelevich et al,Chem. Nat. Compd. (Engl. Transl.), 18, 215 (1982). 18. Mahfouz et al,Eur. J. Med. Chem. Chim. Ther., 34, 551 (1999). 19. Jones et al, Synth.Commun., 16, 1607 (1986). 20. Dittmer and Christy, J. Org. Chem., 26,1324 (1961).

NMR data for Example 83: δ_(H) (400 MHz; CD₃OD) 0.74-0.82 (5H, m),0.90-0.98 (5H, m), 1.10-1.18 (1H, br m), 1.16 (3H, s), 1.28-1.47 (3H,m), 1.44 (3H, s) 1.55-1.72 (3H, m) 1.78-1.84 (1H, m), 2.10-2.35 (5H, m),2.33 (6H, s), 2.68-78 (2H, m), 3.48 (1H, d), 5.14-5.21 (2H, m), 5.70(1H, d), 6.35 (1H, dd).

General Procedure C, for the Preparation of Mutilin14-[N-(alkoxycarbonyl)]carbamate Amide Derivatives

To the acid Intermediate 23 or Intermediate 24 (0.20 mmol) dissolved indry DMF (1 ml) was added 1-hydroxy-7-azabenzotriazole (0.030 g, 0.22mmol) in DMF (1 ml), followed by the amine (0.22 mmol; in the case ofExample 97, 0.44 mmol), [and diethylaminomethyl-polystyrene (0.40 mmol)in the case of amine hydrochlorides] and, after 30 min,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.042 g,0.22 mmol). The mixture was stirred under argon for 24 h, water (1 ml)and saturated sodium hydrogen carbonate solution (1 ml) were added, andthe mixture filtered through hydromatrix gel and washed through withethyl acetate. The filtrate was concentrated in vacuo then purified byflash column chromatography on silica gel, eluting with 20-100% ethylacetate in petroleum ether then 0-20% methanol in dichloromethane togive the title compounds.

Examples 91-99

Examples 91-99 were prepared by general procedure C.

Example Acid R¹R²N— X MW Yield LC/MS (ES+) 91 Int 23 MeNH H 478 81% 501[MNa]⁺, 100% 92 Int. 23 Me₂N H 492 78% 515 [MNa]⁺, 100% 93 Int. 23MeO(CH₂)₂NH H 522 72% 545 [MNa]⁺, 100% 94 Int. 23 NCCH₂NH H 503 68% 526[MNa]⁺, 100% 95 Int. 23 MeONH H 494 72% 517 [MNa]⁺, 100% 96 Int 23

H 534 85% 557 [MNa]⁺, 100% 97 Int. 23

H 533 45% 534, [MH]⁺ 100% 98 Int. 23

H 555 78% 556 [MH]⁺, 100% 99 Int. 24 MeNH Me 506 61% 529 [MNa]⁺, 100%All amines were commercially available; that for Example 95 was used asthe hydrochloride salt.General Procedure D, for the Preparation of Mutilin(2S)-hydroxy-14-[N-(alkoxycarbonyl)]carbamates

(a) (2S)-2-Dichloroacetoxy-11-O-trifluoroacetyl-mutilin 14-chloroformateThe title as prepared as in the method of Example 12 of patentPCT/EP96/05874 from (2)-2-dichloroacetoxy-11-O-trifluroacetyl-mutilin.

(b) Mutilin (2S)-hydroxy-14-[N-(alkoxycarbonyl)]carbamates(2S)-2-Dichloroacetoxy-11-O-trifluoroacetyl-mut 14-chloroformate (0.242g, 0.40 mmol) was dissolved in dry dichloromethane (2.5 ml) and added tosilver cyanate (0.09 g, 0.60 mmol). To this stirred suspension was addedpyridine (0.008 g, 0.10 mmol) in dry dichloromethane (0.1 ml). After 1.5min the appropriate alcohol in dry dichloromethane or DMF was added. Themixture was stirred for 3.5 h, then treated with water (0.1 ml), andethyl acetate added (10 ml). This mixture was then filtered throughhydromatrix gel and kieselguhr. After evaporation the residue wasdissolved in ethanol (8 ml) and saturated aqueous sodium hydrogencarbonate (8 ml) added. The mixture was stirred for 6 h, then filteredand the filtrate extracted with ethyl acetate. The filtered solid wasslurried in the aqueous phase and extracted with ethyl acetate. Thecombined organic extracts were washed with brine, dried and evaporated.The residue was purified by flash column chromatography on silica gel,eluting with 20-100% ethyl acetate in petroleum ether then 0-10%methanol in dichloromethane, or by reverse phase HPLC eluting withacetonitrile in water (with TFA 0.1%) to give the title compounds.

Examples 100 and 101

Examples 100 and 101 were prepared by general procedure D.

Example R Alcohol MW Yield LC/MS 100

C 467 74% 490 (ES+) [MNa]⁺ 100% 101

C 544 33% 545 (ES+) [MH]⁺ 100%General Procedure E, for Reduction of the 19,20 Double Bond

To the starting material (0.05 mmol) in THF (2 ml) under argon was added10% palladium on carbon (20 mg) and the mixture treated with hydrogengas at 1 atm with shaking for 2 h. The reaction mixture was filteredthrough kieselguhr, and the filter pad washed well with THF. Thecombined filtrates were evaporated to dryness, in general nopurification was necessary.

Examples 102-105

Examples 102-105 were prepared by general procedure E.

Example Reactant R¹ R² MW Yield LC/MS 102 Ex. 3

OH 469 90% 468 (APCI−) [M − H]⁻ 100% 103 Ex. 7

H 466 95% 465 (APCI−) [M − H]⁻ 100% 104 Ex. 27

H 506 99% 505 (APCI−) [M − H]⁻ 100% 105 Ex. 34

H 478 85% 479 (APCI+) [MH]⁺ 100%

Example 106 Mutilin14-[N-(1-methanesulfonylpyrrolidin-3-(S)-yloxycarbonyl)]carbamate

To the compound of Example 45 (0.049 g, 0.1 mmol) dissolved in drydichloromethane (2 ml) under argon was added pyridine (0.012 ml) and asolution of methanesulfonyl chloride (0.0135 g, 0.12 mmol) in drydichloromethane (1 ml). The reaction was stirred at 20° C. for 20 h,then ethyl acetate (10 ml) and 1 M hydrochloric acid (1 ml) were added.The phases were separated, and the aqueous layer extracted with furtherethyl acetate (10 ml). The combined organic layers were dried by passagethrough a hydromatrix gel column, evaporated to dryness and purified bychromatography on a Biotage Quad 3 eluting with 0-13% methanol indichloromethane. The product containing fractions were combined andevaporated to dryness to give the title product, as a white foam (0.037g, 66%).

Related derivatives were prepared by the same method using the amineExample 45 or 57 and using methanesulfonyl chloride/pyridine, sulfamoylchloride/triethylamine or 2 equivalents of trimethylsilyl isocyanate.

Example Amine R MW Yield LC/MS 106 Ex. 45

554 66% 553 (ES−) [M − H]⁻ 100% 107 Ex. 57

584 72% 583 (ES−) [M − H]⁻ 100% 108 Ex. 57

585 24% 584 (ES−) [M −H]⁻ 100% 109 Ex. 57

549 23% 548 (ES−) [M − H]⁻ 100% 110 Ex. 45

519 44% 518 (ES−) [M − H]⁻ 100%

Example 111 Mutilin14-[N-(1-glycylpyrrolidin-3(R)-yloxycarbonyl)]carbamate

To the compound of Example 44 (0.05 g, 0.105 mmol) dissolved in dry DMF(0.5 ml) under argon was added Boc-glycine (0.019 g), HOAT (0.014 g) andEDAC (0.021 g). The reaction was stirred at 20° C. for 20 h, andconcentrated by evaporation under reduced pressure. The residue wastaken up in ethyl acetate and water (10 ml, 1 ml), the organic phasewashed with saturated aqueous sodium hydrogen carbonate, and the aqueousphases re-extracted with ethyl actate. The combined organic phases weredried and evaporated to low volume under reduced pressure. The residuewas treated with trifluoroacetic acid (4 ml) at 20° C. for 15 min, thenevaporated to dryness under reduced pressure and purified bychromatography on silica gel eluting with 0-10% 2M methanolic ammonia indichloromethane to give the title product as a white foam (0.041 g,77%).

Further products were made from the products of Examples 44, 45 and 57using the same procedure but substituting the appropriate carboxylicacid for Boc-glycine and omitting the TFA treatment.

Example Amine R MW Yield LC/MS 111 Ex. 44

533 77% 532 (ES−) [M − H]⁻ 100% 112 Ex. 45

547 80% 546 (ES−) [M − H]⁻ 100% 113 Ex. 44

570 60% 593 (APCI+) [MNa]⁺ 15% 114 Ex. 57

577 65% 576 (ES−) [M − H]⁻ 100% 115 Ex. 57

626 46% 625 (ES−) [M − H]⁻ 100%

Example 116 Mutilin14-[N-(4-amino-5-carboxamidopyrimidin-2-ylmethoxycarbonyl)]carbamate

The compound of Example 37 (0.1 g, 0.185 mmol) was treated with conc.HCl (20 ml) for 4 h. The reaction was evaporated to dryness underreduced pressure and the residue purified by chromatography on silicagel eluting with 2-6% 2M methanolic ammonia in dichloromethane to giverecovered starting material, (0.04 g) and the title product as a paleyellow foam (0.022 g, 35% based on recovered starting material), LC/MS(APCI+) 558 [MH]⁺ 100%.

Example 117 Mutilin 14-[N-(methylsulfonylmethoxycarbonyl)]carbamate

To the compound of Example 89 (0.052 g, 0.11 mmol) in dichloromethane (5ml) at 0° C. was added meta-chloroperbenzoic acid (MCPBA, 0.035 g) andthe mixture stirred at 0° C. for 30 min and a further 30 min at 20° C. Afurther 0.005 g MCPBA was added and the reaction stirred at 20° C. for 2h. The reaction mix was washed with saturated aqueous sodium hydrogencarbonate (2 ml), dried and purified by chromatography on silica geleluting with 10-50% ethyl acetate in dichloromethane, to give the titlematerial as a colourless foam (0.032 g, 58%), LC/MS (APCI+) 517 [M.NH₄]⁺100%.

Example 118 Mutilin14-[-(cis-4-carbamoyloxytetrahydrofuran-3-yloxycarbonyl)]carbamate

11-Trifluoroacetylmutilin 14-chloroformate (0.275 g, 0.57 mmol) wasdisolved in dry dichloromethane (5 ml) and added to silver cyanate (0.13g). To this stirred suspension was added pyridine (0.01 5 g). After 1.5min 1,4-anhydroerythritol (0.14 ml) was added. The mixture was stirredfor 3.5 h then treated with water (0.1 ml), ethyl acetate (10 ml) wasadded, and the mixture filtered through hydromatrix gel and kieselguhr.After evaporation the residue was purified by column chromatography onsilica gel, eluting with 40-70% ethyl acetate in petroleum ether, togive11-trifluoroacetylmutilin-14-[N-(cis-4-hydroxytetrahydrofuran-3-yloxycarbonyl)]carbamate,(0.18 g, 57%). This material (0.069 g) in dry THF (2 ml) was treatedwith carbonyl diimidazole (0.13 g) at 20° C. for 1.5 h, then aqueousammonia (20 M, 0.1 ml) was added. After stirring at 20° C. for 2 h, thesolvent was evaporated and the residue triturated withdichloromethane/methanol (9:1, 2 ml). The soluble material was purifiedby chromatography on silica gel eluting with 30-100% ethyl acetate inhexane to give the title material (0.02 g, 30%), LC/MS (ES−) 492[M-CONH2]⁻ 100%, 535 [M−H]⁻ 30%.

Example 119 Mutilin14-[N-((2-amino-3-carbamoylpyrazin-5-yl)methyloxycarbonyl)]carbamate

(a) 5-Acetoxymethyl-2-bis(tert-butoxycarbonyl)amino-3-cyanopyrazine5-Acetoxymethyl-2-amino-3-cyanopyrazine (0.46 g, 2.4 mmol) was suspendedin dichloromethane (18 ml), then triethylamine (0.81 ml, 5.8 mmol) addedfollowed by di-t-butyldicarbonate (1.2 g, 5.5 mmol) and DMAP (few mgs).The mixture was stirred for 1 h, then washed with water and brine, driedand evaporated to give the crude title compound as a pale brown oilysolid (1.05 g, q); NMR (CDCl₃) 1.47 (18H, s), 2.20 (3H, s), 5.32 (2 h,s), and 8.73 (1H, s). This material was used without furtherpurification.

(b) 2-Bis(tert-butoxycarbonyl)amino-3-cyano-5-(hydroxymethyl)pyrazineThe crude product from (a) was dissolved in dioxane (10 ml) and water (2ml) then sodium hydroxide solution (2M, 1.3 ml, 2.6 mmol) added. Themixture was stirred for 5 h, diluted with ethyl acetate, washed withdilute sodium chloride solution then brine, dried and evaporated to givethe crude product as a brown gum (0.95 g). This material waschromatographed, eluting with 0-35% ethyl acetate in petroleum ether, togive the required product contaminated with an impurity(5-acetoxymethyl-2-tert-butoxycarbonylamino-3-cyanopyrazine) (0.172 g).

(c) Mutilin14-[N-((2-amino-3-carbamoylpyrazin-5-yl)methyloxycarbonyl)]carbamateImpure 2-bis(tert-butoxycarbonyl)amino-3-cyano-5-(hydroxymethyl)pyrazine(0.16 g, ˜0.22 mmol) was then reacted as in general method A. During thetreatment with concentrated hydrochloric acid the cyano group washydrolysed to the primary amide and the tert-butoxycarbonyl groupsremoved to give the title compound as a pale yellow foam (0.026 g, 21%);LC/MS (ES+) 580 (MNa+), 100%.

Example 120 Mutilin14-[N-(2-(4-formylpiperazin-1-yl)ethyloxycarbonyl)]carbamate

To the compound of Example 65 (0.13 g, 0.25 mmol) dissolved in drydimethylformamide (2 ml) under argon was added formic acid (0.044 ml,1.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(0.114 g, 0.6 mmol) and 1-hydroxy-7-azabenzotriazole (0.041 g, 0.3mmol). The mixture was stirred for 6 h then diluted with ethyl acetateand aqeous sodium hydrogen carbonate (1 ml), passed through hydromatrixgel, and evaporated. The crude product was purified by chromatography togive the title compound as a white foam (0.104 g, 63%); LC/MS (ES−) 546[M−H]⁻, 100%.

Example 121 Mutilin14-[N-(2-(4-sulphamoylpiperazin-1-yl)ethyloxycarbonyl)]carbamate

To the compound of Example 65 (0.104 g, 0.2 mmol) dissolved in drydichloromethane (2 ml) under argon was added triethylamine (0.031 ml,0.22 mmol) followed by a solution of sulphamoyl chloride (0.024 g, 0.21mmol) in dry dichloromethane (1 ml). Stirred for 2 h then furtherportions of reagents added. After a further 2 h the mixture waschromatographed to give the title compound as an off-white foam (0.030g, 25%); LC/MS (ES−) 597 [M−H]⁻, 100%.

Example 122 Mutilin14-[N-(((3R,3aR,6S,6aR)-6-amino-hexahydrofuro[3,2-b]furan-3-yl)oxycarbonyl)]carbamate

Mutilin14-[N-(((3R,3aR,6S,6aR)-6-azido-hexahydrofuro[3,2-b]furan-3-yl)oxycarbonyl)]carbamate(0.56 g, 1 mmol) was dissolved in tetrahydrofuran (14 ml) thentriphenylphosphine (0.524 g, 2 mmol) and water (0.3 ml) added. Themixture was stirred for 43 h then evaporated and chromatographed to givethe title compound as a white foam (0.294 g, 55%); LC/MS (ES−) 533[M−H]⁻, 100%.

Example 123 Mutilin14-[N-(1-cyanomethylpyrrolidin-3-(R)-yloxycarbonyl)]carbamate

To a solution of the compound of Example 44 (95 mg, 0.2 mmol) indichloromethane (1 ml) was added bromoacetonitrile (18 ul, 0.25 mmol)followed by triethylamine (35 ul, 0.25 mmol) and the solution wasstirred at RT for 3 h. The solution was partitioned between saturatedaqueous sodium hydrogen carbonate and dichloromethane, and the organiclayer was dried (MgSO₄) and evaporated. The residue was purified bycolumn chromatography on silica gel eluting with 0.5% ‘2 M ammonia inmethanol’ in ethyl acetate. The product was triturated with ether-hexaneto afford the title compound as a white solid (78 mg, 76%); LC/MS(APCI+) 538 [MH]⁺, 100%.

Examples 124 and 125

Examples 124 and 125 were prepared by general procedure A. The aqueouswork-up was omitted and the residue was purified by columnchromatography on silica gel eluting with 0-15% 2M methanolic ammonia indichloromethane.

Example R Alcohol MW Yield LC/MS 124

Int 25 492 12% 491 (APCI−) [M − H]⁻ 100% 125

Ref 21, # 490 59% 491 (ES+) [M + H]⁺ 100% # Starting alcohol is N-Bocderivative;Reference 21: Hodgson et al, Tetrahedron Lett.; 39, 3357 (1998).

Example 126 Mutilin14[N-(5-(S)-cyanopyrrolidin-3-(R)-yloxycarbonyl)]carbamate

a) N-o-Nitrophenylsulphenyl trans-4-hydroxy-L-proline Totrans-4-hydroxy-L-proline (6.0 g, 45.8 mmol) in dioxan (60 ml) and 2 Msodium hydroxide (23 ml) was added portionwise o-nitrobenzenenesulfenylchloride (9.5 g, 50 mmol) with simultaneous addition of 2 M sodiumhydroxide (23 ml). The resulting orange reaction mixture was dilutedwith water (200 ml) and washed with diethyl ether. The aqueous solutionwas cooled in ice, acidified to pH 2.5 with c HCl and extracted withethyl acetate. The organic layer was washed with brine, dried (MgSO₄)and evaporated to a gum. Diethyl ether (100 ml) was added and themixture sonicated to give an orange solid which was removed byfiltration and washed with diethyl ether to give the title material,(9.4 g, 72%); NMR (CD3OD) 2.3 (2H, m), 3.1 (1H, bs), 3.7 (1H, bs), 4.16(1H, t), 4.5 (1H, bs), 7.32 (1H, dt), 7.7 (1H, dt),8.25 (1H, bs),8.27(1H, d).

b) N-o-Nitrophenylsulphenyl trans-4-hydroxy-L-prolinamide ToN-o-nitrophenylsulphenyl trans-4-hydroxy-L-proline (5.68 g, 20 mmol) inDMF was added N-hydroxysuccinimide (2.5 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.9 g).After 75 min, ammonium hydrogen carbonate (2 g) and triethylamine (2.8ml) was added and the mixture stirred for 2.5 h. Water (20 ml) was addedand the mixture evaporated to low volume under reduced pressure. To theresidue was added 5% methanol in dichloromethane and the insoluble solidremoved by filtration. The filtrate was purified by chromatography onsilica gel eluting with 0-12% (2 M methanolic ammonia) indichloromethane to give the title compound, (5.1 g) which contained atrace of N-hydroxysuccinamide; NMR (CD₃OD) 2.2 (2H, m), 3.2. (1H, bd),3.8 (1H, bd), 4.15 (1H, t), 4.5 (1H, bm), 7.3 (1H, dt), 7.7 (1H,dt),8.25 (1H, bd), 8.25 (1H, d).

c) 2-(S)-Cyano-4-(R)-hydroxy-1-o-nitrophenylsulphenylpyrrolidine ToN-o-nitrophenylsulphenyl trans-4-hydroxy-L-prolinamide (0.566 g, 2 mmol)in THF (5 ml) at 0° C. under argon was added triethylamine (1.7 ml) andtrifluoroacetic acid anhydride (TFAA; 0.85 ml). After 45 min furtherportions of added triethylamine (1.7 ml) and TFAA (0.85 ml) were added.After a further 45 min water (20 ml) was added, the mixture extractedwith diethyl ether and the organic solution dried and evaporated. Theresidue was purified by chromatography on silica to give the titlematerial as a pale yellow gum, (0.41 g, 77%); NMR (CDCl₃) 1.9 (1H, bs),2.5 (2H, bm), 3.2 (1H, bm), 3.8 (1H, bs), 4.5 (1H, t), 4.65 (1H, bs),7.35 (1H, dt), 7.65 (1H, dt), 8.0 (1H, bm), 8.3 (1H, dd).

d) Mutilin 14-[N-(5-(S)-cyanopyrrolidin-3-(R)-yloycarbonyl)]carbamate2-(S)-Cyano-4-(R)-hydroxy-1-o-nitrophenylsulphenylpyrrolidine (0.133 g,0.5 mmol) was subjected to general procedure B, to give mutilin14-[N-(2-(S)-cyano-1-o-nitrophenylsulphenylpyrrolidin-4-(R)-yloxycarbonyl)]carbamate,(0.141 g). This material was dissolved in dichloromethane (5 ml) andtreated with 1 M HCl in diethyl ether (1.5 ml)

After 10 min, the precipitated material was collected and purified bychromatography on silica gel eluting with 0-10% (2M methanolic ammonia)in dichloromethane to give the title compound as a pale yellow foam, (50mg, 20%); LC/MS (ES−) 500 (M−H⁻), 30%.

Example 127 19,20-Dihydromutilin14-[N-(trans-3-aminocyclopentyloxycarbonyl)]carbamate

trans-3-Benzyloxycarbonylaminocyclopenanol (Gregson et al, WO 9417090,1994) was subjected to general procedure B on a 0.226 mmol scale, togive mutilin14-[N-(trans-3-benzyloxycarbonylaminocyclopentyloxycarbonyl)]carbamateas a colourless gum (0.07 g, 49%); NMR (CDCl₃) 0.77 (3H, d), 0.87 (3H,d), 1.1-1.9 (19H, m), 2.0-2.4 (7H, m), 3.5 (1H, dd), 4.18 (1H, m), 4.7(1H, bs). 5.1 (2H, bs), 5.2 (2H, m), 5.37 (1H, d), 5.75 (1H, d), 6.52(1H, d), 6.1 (1H, bd), 7.3 (5H, bs). To this material in THF (7 ml)under argon was added 10% palladium on carbon (50 mg) and the mixturetreated with hydrogen gas at 1 atm with stirring for 2 h. The reactionmixture was filtered through kieselguhr, and the filter pad washed wellwith THF. The combined filtrates were evaporated to dryness and purifiedby column chromatography on silica gel eluting with 0-10% 2 M methanolicammonia in dichloromethane to give the title compound as a colourlessfoam, (0.037 g, 67%) LC/MS (ES−) 491 (M−H⁻), 100%.

Example 128 Mutilin14-[N-(1-carbamoylmethylazetidin-3-yloxycarbonyl)]-carbamate

To the compound of Example 43 (0.075 g, 0.15 mmol) and bromoacetamide(0.025 g, 0.18 mmol) dissolved in dry dichloromethane (2 ml) under argonwas added triethylamine (0.052 ml, 0.37 mmol). The reaction was stirredfor 2.5 h. Purification by flash chromatography, eluting with 0-7% 2 Mammonia in methanol in dichloromethane, gave the required productcontaminated with triethylamine hydrochloride. This material was furtherpurified by the use of a SCX cartridge. Washing with methanol theneluting with 0.4-0.8 M ammonia in methanol gave the title compound as awhite foam (0.070 g, 91%); LC/MS (ES+) 520 (MH⁺), 100%.

Example 129 Mutilin14-[N-(1-cyanomethylazetidin-3-yloxycarbonyl)]-carbamate

To the compound of Example 43 (0.075 g, 0.15 mmol) and bromoacetonitrile(0.022 g, 0.18 mmol) dissolved in dry dichloromethane (2 ml) under argonwas added triethylamine (0.052 ml, 0.37 mmol). After stirring for 3.5 hfurther portions of bromoacetonitrile and triethylamine were added, andthe mixture stirred for another 1 h. The mixture was purified by flashchromatography, eluting with 0-4% 2M ammonia in methanol indichloromethane, to give the title compound as a white foam (0.063 g,83%); LC/MS (ES+) 524 (MNa⁺), 100%.

Example 130 Mutilin 14-[N-(1-aminoazetidin-3-yloxycarbonyl)]-carbamate

To the compound of Example 43 (0.499 g, 1 mmol) dissolved in drydichloromethane (20 ml) was added diisopropylethylamine (0.191 ml, 1.1mmol). The solution was cooled in an ice bath thenN-tert-butoxycarbonyl-3-(4-cyanophenyl)oxaziridine (0.271 g, 1.1 mmol)in dry dichloromethane (5 ml) added dropwise. The mixture was stirredfor 1.5 h cold and 0.5 h at RT. The volume of the solution was reducedand it was then purified by flash chromatography. Elution with 20-50%ethyl acetate in petroleum ether gave the impure N-tert-butoxycarbonylprotected compound (0.446 g). This material was dissolved intrifluoroacetic acid (5 ml) and stirred for 0.5 h. Evaporation gave awhite foam which was purified by flash chromatography, eluting with 0-6%2M ammonia in methanol in dichloromethane, to give the impure product.Further flash chromatography, eluting with 46% methanol indichloromethane, gave the pure title compound as a white foam (0.203 g,43%); LC/MS (ES−) 476 [M−H]⁻, 100%.

Biological Data

Compounds of the present invention were assessed for anti-bacterialactivity in a conventional MIC assay against a range of pathogenicorganisms.

Examples 1 to 130 were found to have MICs≦4 ug/ml against Staphylococcusaureus Oxford, Streptococcus pneumoniae 1629, Moraxella catarrhalis1502, and Haemophilius influenzae Q1.

1. A compound of formula (IA) or (IB):

in which: R¹ is optionally substituted C₍₁₋₆₎alkyl or C₍₃₋₆₎cycloalkylor optionally substituted heterocyclyl; R² is vinyl or ethyl; R³ is H,OH or F, and R⁴ is H, or R³ is H and R⁴ is F, and R⁵ and R⁶ togetherform an oxo group; or R³ and R⁴ is each H, R⁵ is OH or H and R⁶ is H, orR⁵ is H and R⁶ is OH or H; or a pharmaceutically acceptable saltthereof.
 2. A compound or salt as claimed in claim 1 in which R¹ isselected from C₍₁₋₆₎alkyl or C₍₃₋₆₎cycloalkyl optionally substituted byup to four substituents selected from aryl, heteroaryl, heterocyclyl,(C₁₋₆)alkoxy, (C₁₋₆)alkylthio, aryl(C₁₋₆)alkoxy, aryl(C₁₋₆)alkylthio,amino, mono- or di-(C₁₋₆)alkylamino, cycloalkyl, cycloalkenyl, carboxyand esters thereof, carbamoyl, ureido, guanidino, (C₁₋₆)alkylguanidino,amidino, (C₁₋₆)alkylamidino, (C₁₋₆)acyloxy, azido, hydroxy, halogen,substituted aryl, substituted heteroaryl, substituted heterocyclyl,substituted amino, carbamoyloxy, ureido substituted by (C₁₋₆)alkyl,(C₁₋₆)alkylsulphonyl, mono- or di-(C₁₋₆)alkylamino(C₁₋₆)alkyl, andcarbamoyl substituted by 1 or 2 substituents which may be the same ordifferent selected from (C₁₋₆)alkyl, (C₁₋₆)alkoxy,(C₁₋₆)alkoxy(C₁₋₆)alkyl, cyano(C₁₋₆)alkyl and heteroaryl(C₁₋₆)alkyl, orcarbamoyl substituted by 2 substituents which together with the nitrogenatom to which they are attached form a heterocyclyl ring; andheterocyclyl optionally substituted by up to three substituents selectedfrom oxo, (C₁₋₆)alkylimino, halogen, (C₁₋₆)alkyl, aryl, aryl(C₁₋₆)alkyl,(C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl, halo(C₁₋₆)alkyl,aryl(C₁₋₆)alkoxy, hydroxy, nitro, cyano, azido, amino, mono- anddi-N-(C₁₋₆)alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxy,carboxy salts, carboxy esters, carbamoyl, mono- anddi-N-(C₁₋₆)alkylcarbamoyl, (C₁₋₆)alkoxycarbonyl, aryloxycarbonyl,ureido, guanidino, (C₁₋₆)alkylguanidino, amidino, (C₁₋₆)alkylamidino,sulphonylamino, aminosulphonyl, (C₁₋₆)alkylthio, (C₁₋₆)alkylsulphinyl,(C₁₋₆)alkylsulphonyl, heterocyclyl, heteroaryl, heterocyclyl(C₁₋₆)alkyl,heteroaryl(C₁₋₆)alkyl substituted (C₁₋₆)alkyl, substituted amino,amino(C₁₋₆)alkyl, amino(C₁₋₆)alkylcarbonyl, oxamoyl,carbamoyl(C₁₋₆)alkyl, cyano(C₁₋₆)alkyl, acyl, carbamoyloxy,carbamoyloxy(C₁₋₆)alkyl and optionally substituted heteroarylcarbonyl,or two adjacent ring carbon atoms are linked by a (C₃₋₅)alkylene chain,to form a carbocyclic ring.
 3. A compound or salt as claimed in claim 1in which R¹ is C₍₁₋₆₎alkyl optionally substituted by 1, 2 or 3substituents selected from heteroaryl optionally substituted by 1, 2 or3 substituents selected from C₍₁₋₆₎alkyl, hydroxyC₍₁₋₆₎alkyl, amino,aminoC₍₁₋₆₎alkyl, carbamoyl, cyano and hydroxy, heterocyclyl optionallysubstituted by 1, 2 or 3 substituents selected from oxo, C₍₁₋₆₎alkyl,hydroxyC₍₁₋₆₎alkyl, aminosulphonyl, carbamoyl and acyl, aminosubstituted by 1 or 2 substituents which may be the same or differentselected from oxamoyl (optionally substituted on N byhydroxy(C₁₋₆)alkyl), acyl, (C₁₋₆)alkylsulphonyl, (C₁₋₆)alkyl (optionallysubstituted by, for example, hydroxy), and carbamoyl(C₁ 6)alkyl,carbamoyloxy, ureido substituted by C₍₁₋₆₎alkyl, C₍₁₋₆₎alkylsulphonyl,hydroxy, halogen, C₍₁₋₆₎alkylthio, and R⁷R⁸NCO wherein R⁷ and R⁸ whichmay be the same or different is each selected from hydrogen,(C₁₋₆)alkyl, (C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl, cyano(C₁₋₆)alkyl andheteroaryl(C₁₋₆)alkyl or R⁷ and R⁸ together with the nitrogen atom towhich they are attached form a heterocyclyl ring.
 4. A compound or saltas claimed in claim 3 in which R¹ is selected from C₍₁₋₆₎alkylsubstituted by carbamoyloxy and C₍₁₋₆₎alkyl substituted by hydroxy anddi-N-C₍₁₋₆₎alkylamino.
 5. A compound or salt as claimed in claim 1 inwhich R¹ is C₍₃₋₆₎cycloalkyl substituted by amino, carbamoyl ordi-C₍₁₋₆₎alkylaminoC₍₁₋₆₎alkyl.
 6. A compound or salt as claimed inclaim 5 in which R¹ is C₍₃₋₆₎cycloalkyl substituted bydi-C₍₁₋₆₎alkylaminoC₍₁₋₆₎alkyl.
 7. A compound or salt as claimed inclaim 1 in which R¹ is a 4- to 6-membered heterocyclyl ring or two fused5-membered heterocyclyl rings containing 1 or 2 heteroatoms in each ring(for example tetrahydrofuran, 1,3-dioxane, hexahydrofuro[3,2-b]furan,trimethylenesulphide, tetrahydrothiophene, azetidine, pyrrolidine,isoxazolidine or piperidine) optionally substituted by 1, 2 or 3substituents selected from C₍₁₋₆₎alkyl, hydroxy, hydroxy C₍₁₋₆₎alkyl,C₍₁₋₆₎alkylsulphonyl, C₍₁₋₆₎alkoxy, oxo, oxamoyl, carbamoyl, carbamoylC₍₁₋₆₎alkyl, carbamoyloxy, carbamoyloxyC₍₁₋₆₎alkyl,aminoC₍₁₋₆₎alkylcarbonyl amino (optionally substituted by oxamoyl),C₍₁₋₆₎alkoxyimino, azido, cyano, cyano(C₁₋₆)alkyl and heteroarylcarbonyl(optionally substituted by, for example, amino).
 8. A compound or saltas claimed in claim 7 in which R¹ is a 4- or 5-membered heterocyclylring containing one nitrogen atom optionally substituted by oxo.
 9. Acompound or salt as claimed in claim 1 in which R³ and R⁴ are bothhydrogen, and R⁵ and R⁶ together form an oxo group.
 10. A compoundaccording to claim 1 selected from: Mutilin14-[N-(2-carbamoyloxyethoxycarbonyl)]carbamate; Mutilin14-[N-(2-oxopyrrolidin-3-(S)-yloxycarbonyl)]carbamate; Mutilin14-[N-(azetidinyloxycarbonyl)]carbamate hydrochloride; Mutilin14-[N-(pyrrolidin-3-(R)-yloxycarbonyl)]carbamate; Mutilin14-[N-(pyrrolidin-3-(S)-yloxycarbonyl)]carbamate; Mutilin14-[N-(3-dimethylamino-2-hydroxyprop-1-yloxycarbonyl)]carbamate; andMutilin 14-[N-(1-(dimethylaminomethyl)cyclopropoxycarbonyl)]carbamate;or a pharmaceutically acceptable salt thereof.
 11. A method of preparinga compound of formula (IA) or (IB) which process comprises: (a) reactinga chloroformate compound of formula (IIA) or (IIB):

in which: P is hydrogen or an hydroxy-protecting group; R^(2A), R^(3A),R^(4A), R^(5A) and R^(6A) are R², R³, R⁴, R⁵ and R⁶ as defined forformulae (IA) and (IB) or a group convertible to R², R³, R⁴, R⁵ and R⁶respectively; with silver cyanate, in a suitable aprotic solvent, forinstance dichloromethane, followed by treating with an alcohol compoundof formula (a):R^(1A)OH  (III) in which R^(1A) is R¹ as defined for formulae (IA) and(IB) or a group convertible to R¹; in the presence of pyridine, in acarbamate forming reaction, and thereafter, and if so needed; convertingP to hydrogen, and, if necessary, converting an R^(2A), R^(3A), R^(4A),R^(5A) and R^(6A) group to an R², R³, R⁴, R⁵ and R⁶ group; or (b)chloroformate epi-mutilin compound of formula (IIC):

wherein R^(2A) is as hereinbefore defined; with silver cyanate and thena compound of formula (III), as hereinbefore defined; under carbamateforming conditions as hereinbefore described; and then treating theproduct with an acid; and where required or desired converting an R^(1A)group to an R¹ group and an R^(2A) group to an R² group; or (c) aprocess corresponding to (a) or (b) in which the chloroformate componentcomprises R^(1A) and the alcohol component comprises the mutilinnucleus.
 12. A pharmaceutical composition comprising a compound or saltaccording to claim 1 and a pharmaceutically acceptable carrier.
 13. Amethod of treating microbial infections which comprises administering acompound or salt according to claim 1 to a patient in need thereof.